Hepatitis C virus immunogenic compositions and methods of use thereof

ABSTRACT

The present disclosure provides heterodimeric polypeptides comprising: 1) a variant hepatitis C virus (HCV) E2 polypeptide and an HCV E1 polypeptide; 2) a variant HCV E1 polypeptide and an HCV E2 polypeptide; or 3) a variant HCV E1 polypeptide and a variant HCV E2 polypeptide, where the variant HCV E2 polypeptide and/or the HCV E1 polypeptide comprises one or more T cell epitopes, present in an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide. The present disclosure provides nucleic acids encoding a polyprotein that includes E1 and variant E2, E2 and variant E1, or variant E2 and variant E1. The present disclosure provides a method of producing an E1/E2 heterodimer of the present disclosure. The present disclosure provides a method of inducing an immune response in an individual. The present disclosure provides variant E2 polypeptides and variant E1 polypeptides; and nucleic acids encoding same.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/374,403, filed Apr. 3, 2019, now U.S. Pat. No. 10,881,727, which is acontinuation of U.S. patent application Ser. No. 15/574,427, filed Nov.15, 2017, now U.S. Pat. No. 10,300,131, which is a national stage filingunder 35 U.S.C. § 371 of PCT/IB2016/001051, filed Jul. 6, 2016, whichclaims the benefit of U.S. Provisional Patent Application No.62/189,657, filed Jul. 7, 2015, each of which applications isincorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“UALB-027WO_Sequence_Listing_ST25.txt” created on Jul. 5, 2016 andhaving a size of 933 KB. The contents of the text file are incorporatedby reference herein in their entirety.

INTRODUCTION

Hepatitis C virus (HCV) is a blood-borne pathogen that is estimated toinfect 150-200 million people worldwide. Infection by HCV may benon-symptomatic, and can be cleared by patients, sometimes withoutmedical intervention. However, the majority of patients develop achronic HCV infection, which may lead to liver inflammation, scarring,and even to liver failure or liver cancer. In the United States alone,over 3 million people have a chronic infection.

The HCV virion contains a positive-sense single stranded RNA genome ofabout 9.5 kb. The genome encodes a single polyprotein of 3,010 to 3,030amino acids. The structural proteins comprise a core protein forming theviral nucleocapsid and two envelope glycoproteins, E1 and E2.

A vaccine based on the recombinant envelope glycoproteins (rE1E2) from asingle genotype 1a strain (HCV-1) protected chimpanzees from chronicinfection following homologous and heterologous genotype 1a (gt1a) viralchallenge (reviewed in Houghton, M Imnmunol Rev 2011). Antisera from theimmunized chimpanzees were shown to exhibit in vitro cross-neutralizingactivity (Meunier et al. (2011) J. Infect. Dis. 204:1186). A phase Iclinical trial was conducted in human volunteers with a similar antigen(Frey et al. (2010) Vaccine 28:6367). Antisera from selected vaccinatedindividuals were similarly capable of neutralizing chimeric cellculture-derived viruses (HCVcc) expressing the structural proteins ofstrains representing all 7 major HCV genotypes in vitro (Law et al.(2013) PloS One 8:e59776) and to be able to compete with the binding ofnumerous discrete monoclonal antibodies with broad cross-neutralisingactivities (Wong et al. (2014) J. Virol. 88:14278).

There is a need in the art for compositions and methods for inducingimmune responses to HCV.

SUMMARY

The present disclosure provides heterodimeric polypeptides comprising avariant hepatitis C virus (HCV) E2 polypeptide and an HCV E1polypeptide, or comprising a variant HCV E1 polypeptide and an HCV E2polypeptide, or comprising a variant HCV E1 polypeptide and a variantHCV E2 polypeptide, where the variant HCV E2 polypeptide and/or thevariant HCV E1 polypeptide comprises one or more T cell epitopes, e.g.,one or more T cell epitopes present in an HCV polypeptide other than anHCV E1 polypeptide or an HCV E2 polypeptide. The present disclosureprovides nucleic acids encoding a polyprotein that includes E1 andvariant E2, or that includes E2 and variant E1, or that includes variantE2 and variant E1. The present disclosure provides a method of producingan E1/E2 heterodimer of the present disclosure. The present disclosureprovides a method of inducing an immune response in an individual. Thepresent disclosure provides variant E2 polypeptides and variant E1polypeptides; and nucleic acids encoding the variant polypeptides.

The present disclosure provides a heterodimeric polypeptide comprising:a) a variant HCV E2 polypeptide comprising: i) an HCV E2 polypeptide;and ii) a heterologous polypeptide comprising a T-cell epitope presentin an HCV protein other than E1 and E2; and b) an HCV E1 polypeptide. Insome cases, the heterologous polypeptide comprises one or more T cellepitopes present in one or more of: a) an HCV non-structuralpolypeptide-3 (NS3) polypeptide; b) an HCV non-structural polypeptide-2(NS2) polypeptide; c) an HCV non-structural polypeptide-4A (NS4A)polypeptide; d) an HCV non-structural polypeptide-4B (NS4B) polypeptide;e) an HCV non-structural polypeptide-5A (NS5A) polypeptide; f) an HCVnon-structural polypeptide-5B (NS5B) polypeptide; g) an HCV corepolypeptide; and h) an HCV p7 polypeptide. In some cases, a) the HCV E2polypeptide is derived from an HCV of genotype 1, 2, 3, or 7; and b) theHCV E1 polypeptide is derived from an HCV of genotype 1, 2, 3, or 7. Insome cases, the HCV E2 polypeptide comprises an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to an E2 polypeptide depicted in one of FIG. 1A-1C,FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B. In some cases, the heterologouspolypeptide has a length of from about 10 amino acids to about 3000amino acids. In some cases the heterologous polypeptide has a length offrom about 10 amino acids to about 100 amino acids. In some cases theheterologous polypeptide comprises one or more T cell epitopes presentin an HCV NS3 polypeptide. In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least 20% amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1). In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast 20% amino acid sequence identity to the following amino acidsequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ IDNO:2). In some cases, the heterologous polypeptide comprises an aminoacid sequence having at least 20% amino acid sequence identity to thefollowing amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATD ALMTGFTGDFDSVIDCN(SEQ ID NO:3). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least 20% amino acid sequence identity tothe following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4). In somecases, the variant HCV E2 polypeptide comprises, in order fromN-terminus to C-terminus: i) the heterologous polypeptide; and ii) theHCV E2 polypeptide. In some cases, the variant HCV E2 polypeptidecomprises, in order from N-terminus to C-terminus: i) the HCV E2polypeptide; and ii) the heterologous polypeptide. In some cases, theheterologous polypeptide comprises one or more T cell epitopes presentin: a) cholera toxin or toxoid; and/or b) tetanus toxin or toxoid;and/or c) diphtheria toxin or toxoid; and/or d) CRM197. In some cases,the E2 polypeptide and/or the E1 polypeptide lacks a C-terminaltransmembrane domain. In some cases, the modified HCV E2 polypeptide andthe HCV E1 polypeptide are derived from an HCV of the same genotype. Insome cases, the modified HCV E2 polypeptide and the HCV E1 polypeptideare derived from an HCV of different genotypes. In some cases, the HCVE1 polypeptide comprises an amino acid sequence having at least 20%amino acid sequence identity to an E1 polypeptide depicted in FIG.1A-1C, FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B.

The present disclosure provides a nucleic acid comprising nucleotidesequences encoding any one of the heterodimeric polypeptides asdescribed above or elsewhere herein. In some cases, the nucleotidesequence encoding the variant HCV E2 polypeptide and the nucleotidesequence encoding the HCV E1 polypeptide are operably linked to apromoter. The present disclosure provides a recombinant expressionvector comprising the nucleic acid.

The present disclosure provides a heterodimeric polypeptide comprising:a) a variant HCV E1 polypeptide comprising: i) an HCV E1 polypeptide;and ii) a heterologous polypeptide comprising a T-cell epitope presentin an HCV protein other than E1 and E2; and b) an HCV E2 polypeptide. Insome cases, the heterologous polypeptide comprises one or more T cellepitopes present in one or more of: a) an HCV NS3 polypeptide; b) an HCVNS2 polypeptide; c) an HCV NS-4A polypeptide; d) an HCV NS4Bpolypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5B polypeptide; g)an HCV core polypeptide; and h) an HCV p7 polypeptide. In some cases, a)the HCV E2 polypeptide is derived from an HCV of genotype 1, 2, 3, or 7;and b) the HCV E1 polypeptide is derived from an HCV of genotype 1, 2,3, or 7. In some cases, the HCV E1 polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to an E1polypeptide depicted in one of FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, andFIG. 4A-4B. In some cases, the heterologous polypeptide has a length offrom about 10 amino acids to about 3000 amino acids. In some cases, theheterologous polypeptide has a length of from about 10 amino acids toabout 100 amino acids. In some cases, the heterologous polypeptidecomprises one or more T cell epitopes present in an HCV NS3 polypeptide.In some cases, the heterologous polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to thefollowing amino acid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ IDNO:1). In some cases, the heterologous polypeptide comprises an aminoacid sequence having at least 20% amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to thefollowing amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATD ALMTGFTGDFDSVIDCN(SEQ ID NO:3). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least 20% amino acid sequence identity tothe following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4). In somecases, the variant HCV E1 polypeptide comprises, in order fromN-terminus to C-terminus: i) the heterologous polypeptide; and ii) theHCV E1 polypeptide. In some cases, the variant HCV E1 polypeptidecomprises, in order from N-terminus to C-terminus: i) the HCV E1polypeptide; and ii) the heterologous polypeptide. In some cases, theheterologous polypeptide comprises one or more T cell epitopes presentin: a) cholera toxin or toxoid; and/or b) tetanus toxin or toxoid;and/or c) diphtheria toxin or toxoid; and/or d) CRM197. In some cases,the E2 polypeptide and/or the E1 polypeptide lacks a C-terminaltransmembrane domain. In some cases, the modified HCV E2 polypeptide andthe HCV E1 polypeptide are derived from an HCV of the same genotype. Insome cases, the modified HCV E2 polypeptide and the HCV E1 polypeptideare derived from an HCV of different genotypes. In some cases, the HCVE2 polypeptide comprises an amino acid sequence having at least 20%amino acid sequence identity to an E2 polypeptide depicted in FIG.1A-1C, FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B.

The present disclosure provides nucleic acid comprising nucleotidesequences encoding any one of the heterodimeric polypeptides describedabove or elsewhere herein. In some cases, the nucleotide sequenceencoding the variant HCV E2 polypeptide and the nucleotide sequenceencoding the HCV E1 polypeptide are operably linked to a promoter. Thepresent disclosure provides a recombinant expression vector comprisingthe nucleic acid.

The present disclosure provides a heterodimeric polypeptide comprising:a) a variant HCV E1 polypeptide comprising: i) an HCV E1 polypeptide;and ii) a first heterologous polypeptide comprising a T-cell epitopepresent in an HCV protein other than E1 and E2; and b) a variant HCV E2polypeptide comprising: i) an HCV E2 polypeptide; and ii) a secondheterologous polypeptide comprising a T-cell epitope present in an HCVprotein other than E1 and E2. In some cases, the first and the secondheterologous polypeptides comprise one or more T cell epitopes presentin one or more of: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide;c) an HCV NS-4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5Apolypeptide; f) an HCV NS5B polypeptide; g) an HCV core polypeptide; andh) an HCV p7 polypeptide. In some cases, a) the HCV E2 polypeptide isderived from an HCV of genotype 1, 2, 3, or 7; and b) the HCV E1polypeptide is derived from an HCV of genotype 1, 2, 3, or 7. In somecases, the HCV E1 polypeptide comprises an amino acid sequence having atleast 20% amino acid sequence identity to an E1 polypeptide depicted inone of FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B. In somecases, each of the first and the second heterologous polypeptideindependently has a length of from about 10 amino acids to about 3000amino acids. In some cases, each of the first and the secondheterologous polypeptide independently has a length of from about 10amino acids to about 100 amino acids. In some cases, the first and/orthe second heterologous polypeptide comprises one or more T cellepitopes present in an HCV NS3 polypeptide. In some cases, the firstand/or the second heterologous polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to thefollowing amino acid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ IDNO:1). In some cases, the first and/or the second heterologouspolypeptide comprises an amino acid sequence having at least 20% aminoacid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2). Insome cases, the first and/or the second heterologous polypeptidecomprises an amino acid sequence having at least 20% amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATD ALMTGFTGDFDSVIDCN(SEQ ID NO:3). In some cases, the first and/or the second heterologouspolypeptide comprises an amino acid sequence having at least 20% aminoacid sequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4). In somecases, the variant HCV E1 polypeptide comprises, in order fromN-terminus to C-terminus: i) the heterologous polypeptide; and ii) theHCV E1 polypeptide, and wherein the variant HCV E2 polypeptidecomprises, in order from N-terminus to C-terminus: i) the heterologouspolypeptide; and ii) the HCV E2 polypeptide. In some cases, the variantHCV E1 polypeptide comprises, in order from N-terminus to C-terminus: i)the HCV E1 polypeptide; and ii) the heterologous polypeptide, andwherein the variant HCV E2 polypeptide comprises, in order fromN-terminus to C-terminus: i) the HCV E2 polypeptide; and ii) theheterologous polypeptide. In some cases, the first and/or the secondheterologous polypeptide comprises one or more T cell epitopes presentin: a) cholera toxin or toxoid; and/or b) tetanus toxin or toxoid;and/or c) diphtheria toxin or toxoid; and/or d) CRM197. In some cases,the E2 polypeptide and/or the E1 polypeptide lacks a C-terminaltransmembrane domain. In some cases, the modified HCV E2 polypeptide andthe HCV E1 polypeptide are derived from an HCV of the same genotype. Insome cases, the modified HCV E2 polypeptide and the HCV E1 polypeptideare derived from an HCV of different genotypes. In some cases, the HCVE2 polypeptide comprises an amino acid sequence having at least 20%amino acid sequence identity to an E2 polypeptide depicted in FIG.1A-1C, FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B.

The present disclosure provides a nucleic acid comprising nucleotidesequences encoding any one of the heterodimeric polypeptides describedabove or elsewhere herein. In some cases, the nucleotide sequenceencoding the variant HCV E2 polypeptide and the nucleotide sequenceencoding the HCV E1 polypeptide are operably linked to a promoter. Thepresent disclosure provides a recombinant expression vector comprisingthe nucleic acid.

The present disclosure provides a composition comprising: a) aheterodimeric polypeptide as described above or elsewhere herein; and b)a pharmaceutically acceptable excipient. In some cases, thepharmaceutically acceptable excipient comprises an adjuvant. In somecases, the adjuvant is MF59, alum, poly(DL-lactide co-glycolide), a CpGoligonucleotide, or keyhole limpet hemocyanin.

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding an HCV E1-E2 polyprotein comprising: a) an HCV E1polypeptide; b) an HCV E2 polypeptide; and c) a heterologous polypeptidethat comprises one or more T cell epitopes present in an HCV polypeptideother than an HCV E1 polypeptide or an HCV E2 polypeptide. In somecases, the HCV E1 polypeptide comprises an amino acid sequence having atleast 20% amino acid sequence identity to an E1 polypeptide depicted inFIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B. In some cases, theHCV E2 polypeptide is derived from an HCV of genotype 1, 2, 3, or 7. Insome cases, the HCV E2 polypeptide comprises an amino acid sequencehaving at least 20% amino acid sequence identity to an E2 polypeptidedepicted in FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B. In somecases, the HCV E2 polypeptide is derived from an HCV of genotype 1, 2,3, or 7. In some cases, the HCV E2 polypeptide and the HCV E1polypeptide are of the same genotype. In some cases, the HCV E2polypeptide and the HCV E1 polypeptide are of different genotypes. Insome cases, the heterologous polypeptide has a length of from about 10amino acids to about 3000 amino acids. In some cases, the heterologouspolypeptide has a length of from about 10 amino acids to about 100 aminoacids. In some cases, the E1-E2 polyprotein comprises, in order fromN-terminus to C-terminus: i) the HCV E1 polypeptide; ii) theheterologous polypeptide; and iii) the HCV E2 polypeptide. In somecases, the E1-E2 polyprotein comprises, in order from N-terminus toC-terminus: i) the HCV E1 polypeptide; ii) the HCV E2 polypeptide; andiii) the heterologous polypeptide. In some cases, the E1-E2 polyproteincomprises, in order from N-terminus to C-terminus: i) the heterologouspolypeptide; ii) the HCV E1 polypeptide; and iii) the HCV E2polypeptide. In some cases, the E1-E2 polyprotein comprises, in orderfrom N-terminus to C-terminus: i) the HCV E1 polypeptide; ii) theheterologous polypeptide; and iii) the HCV E2 polypeptide. In somecases, the nucleic acid comprises a nucleotide sequence encoding animmunoglobulin Fc polypeptide. In some cases, the polypeptide encoded bythe nucleic acid comprises, in order from N-terminus to C-terminus: i)the E1 polypeptide; ii) the Fc polypeptide; iii) the heterologouspolypeptide; and iv) the HCV E2 polypeptide. In some cases, thepolypeptide encoded by the nucleic acid comprises a proteolyticallycleavable linker interposed between the Fc polypeptide and theheterologous polypeptide. In some cases, the polypeptide encoded by thenucleic acid comprises, in order from N-terminus to C-terminus: i) theE1 polypeptide; ii) the Fc polypeptide; iii) the HCV E2 polypeptide; andiv) the heterologous polypeptide. In some cases, a proteolyticallycleavable linker is interposed between the Fc polypeptide and the HCV E2polypeptide. In some cases, a) the proteolytically cleavable linkercomprises the sequence LEVLFQGP (SEQ ID NO:5), wherein cleavage occursbetween the glutamine and the glycine; b) the proteolytically cleavablelinker comprises the sequence ENLYTQS (SEQ ID NO:6), wherein cleavageoccurs between the glutamine and the serine; c) the proteolyticallycleavable linker comprises the sequence DDDDK (SEQ ID NO:7), whereincleavage occurs immediately C-terminal to the lysine residue; or d) theproteolytically cleavable linker comprises the sequence LVPR (SEQ IDNO:8). In some cases, the heterologous polypeptide comprises one or moreT cell epitopes present in one or more of: a) an HCV NS3 polypeptide; b)an HCV NS2 polypeptide; c) an HCV NS4A polypeptide; d) an HCV NS4Bpolypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5B polypeptide; g)an HCV core polypeptide; and h) an HCV p7 polypeptide. In some cases,the heterologous polypeptide comprises one or more T cell epitopespresent in: a) cholera toxin or toxoid; and/or b) tetanus toxin ortoxoid; and/or c) diphtheria toxin or toxoid; and/or d) CRM197. In somecases, the heterologous polypeptide comprises one or more T cellepitopes present in an HCV NS3 polypeptide. In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast 20% amino acid sequence identity to the following amino acidsequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1). In some cases,the heterologous polypeptide comprises an amino acid sequence having atleast 20% amino acid sequence identity to the following amino acidsequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ IDNO:2). In some cases, the heterologous polypeptide comprises an aminoacid sequence having at least 20% amino acid sequence identity to thefollowing amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATD ALMTGFTGDFDSVIDCN(SEQ ID NO:3). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least 20% amino acid sequence identity tothe following amino acid sequence:

(SEQ ID NO: 4) VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF.

The present disclosure provides a recombinant expression vectorcomprising a nucleic acid as described above or elsewhere herein. Insome cases, the nucleotide sequence is operably linked to a promoter. Insome cases, the promoter is functional in a eukaryotic cell. The presentdisclosure provides a genetically modified in vitro host cell comprisinga nucleic acid as described above or elsewhere herein, or therecombinant vector. In some cases, the host cell is a eukaryotic cell.In some cases, the host cell is a mammalian cell.

The present disclosure provides a method of making a variant HCV E1-E2heterodimer, the method comprising: a) contacting a lysate of agenetically modified host cell described above, or elsewhere herein,with an Fc-binding polypeptide immobilized on an insoluble support,wherein the encoded HCV E1-E2 heterodimer comprises an Fc polypeptide,and wherein the HCV E1-E2 heterodimer present in the lysate binds to theimmobilized Fc-binding polypeptide, generating an immobilized HCV E1-E2heterodimer; and b) contacting the immobilized HCV E1-E2 heterodimerwith an enzyme that cleaves the proteolytic cleavage site interposedbetween the Fc polypeptide and the heterologous polypeptide, therebyreleasing the variant HCV E1-E2 heterodimer. In some cases, the releasedvariant HCV E1-E2 heterodimer is at least 50% pure. In some cases, theFc binding polypeptide is Protein A, Protein G, or a Protein A/G fusion.In some cases, a) the proteolytically cleavable linker comprises thesequence LEVLFQGP (SEQ ID NO:5), wherein cleavage occurs between theglutamine and the glycine; b) the proteolytically cleavable linkercomprises the sequence ENLYTQS (SEQ ID NO:6), wherein cleavage occursbetween the glutamine and the serine; c) the proteolytically cleavablelinker comprises the sequence DDDDK (SEQ ID NO:7), wherein cleavageoccurs immediately C-terminal to the lysine residue; or d) theproteolytically cleavable linker comprises the sequence LVPR (SEQ IDNO:8). In some cases, the enzyme is human rhinovirus 3C protease, atobacco etch virus protease, an enterokinase, or thrombin. In somecases, the method further comprises subjecting a composition comprisingthe released HCV E1-E2 heterodimer to hydroxyapatite chromatography.Hydroxyapatite chromatography can be carried out as described in, e.g.,Mazzocca et al. (2005) J. Biol. Chem. 280:11329.

The present disclosure provides a variant HCV E2 polypeptide comprising:a) an HCV E2 polypeptide; and b) a heterologous polypeptide thatcomprises one or more T cell epitopes present in an HCV protein otherthan E1 and E2. In some cases, the HCV E2 polypeptide is derived from anHCV of genotype 1, 2, 3, or 7. In some cases, the HCV E2 polypeptidecomprises an amino acid sequence having at least 20% amino acid sequenceidentity to an E2 polypeptide depicted in one of FIG. 1A-1C, FIG. 2A-2C,FIG. 3A-3C, and FIG. 4A-4B. In some cases, the heterologous polypeptidehas a length of from about 10 amino acids to about 3000 amino acids. Insome cases, the heterologous polypeptide has a length of from about 10amino acids to about 100 amino acids. In some cases, wherein theheterologous polypeptide comprises one or more T cell epitopes presentin one or more of: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide;c) an HCV NS-4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5Apolypeptide; f) an HCV NS5B polypeptide; g) an HCV core polypeptide; andh) an HCV p7 polypeptide. In some cases, the HCV E2 polypeptide lacks aC-terminal transmembrane domain. In some cases, the heterologouspolypeptide comprises one or more T cell epitopes present in: a) choleratoxin or toxoid; and/or b) tetanus toxin or toxoid; and/or c) diphtheriatoxin or toxoid; and/or d) CRM197. In some cases, the heterologouspolypeptide comprises one or more T cell epitopes present in an HCV NS3polypeptide. In some cases, the heterologous polypeptide comprises anamino acid sequence having at least 20% amino acid sequence identity tothe following amino acid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ IDNO:1). In some cases, the heterologous polypeptide comprises an aminoacid sequence having at least 20% amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to thefollowing amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATD ALMTGFTGDFDSVIDCN(SEQ ID NO:3). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least 20% amino acid sequence identity tothe following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4). In somecases, the variant HCV E2 polypeptide comprises, in order fromN-terminus to C-terminus: i) the heterologous polypeptide; and ii) theHCV E2 polypeptide. In some cases, the variant HCV E2 polypeptidecomprises, in order from N-terminus to C-terminus: i) the HCV E2polypeptide; and ii) the heterologous polypeptide. In some cases, thevariant HCV E2 polypeptide comprises an immunoglobulin Fc polypeptide.In some cases, the variant HCV E2 polypeptide comprises, in order fromN-terminus to C-terminus: i) the Fc polypeptide; ii) the heterologouspolypeptide; and iii) the HCV E2 polypeptide. In some cases, the variantHCV E2 polypeptide comprises a proteolytic cleavage site interposedbetween the Fc polypeptide and the heterologous polypeptide. In somecases, the proteolytic cleavage site comprises the sequence LEVLFQGP(SEQ ID NO:5), wherein cleavage occurs between the glutamine and theglycine.

The present disclosure provides a variant HCV E1 polypeptide comprising:a) an HCV E1 polypeptide; and b) a heterologous polypeptide thatcomprises one or more T cell epitopes. In some cases, the HCV E1polypeptide is derived from an HCV of genotype 1, 2, 3, or 7. In somecases, the HCV E1 polypeptide comprises an amino acid sequence having atleast 20% amino acid sequence identity to an E1 polypeptide depicted inone of FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B. In somecases, the heterologous polypeptide has a length of from about 10 aminoacids to about 3000 amino acids. In some cases, the heterologouspolypeptide has a length of from about 10 amino acids to about 100 aminoacids. In some cases, the HCV E1 polypeptide lacks a transmembranedomain. In some cases, the heterologous polypeptide comprises one ormore T cell epitopes present in one or more of: a) an HCV NS3polypeptide; b) an HCV NS2 polypeptide; c) an HCV NS-4A polypeptide; d)an HCV NS4B polypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5Bpolypeptide; g) an HCV core polypeptide; and h) an HCV p7 polypeptide.In some cases, the heterologous polypeptide comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) CRM197. Insome cases, the heterologous polypeptide comprises one or more T cellepitopes present in an HCV NS3 polypeptide. In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast 20% amino acid sequence identity to the following amino acidsequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1). In some cases,the heterologous polypeptide comprises an amino acid sequence having atleast 20% amino acid sequence identity to the following amino acidsequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ IDNO:2). In some cases, the heterologous polypeptide comprises an aminoacid sequence having at least 20% amino acid sequence identity to thefollowing amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATD ALMTGFTGDFDSVIDCN(SEQ ID NO:3). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least 20% amino acid sequence identity tothe following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4). In somecases, the variant HCV E1 polypeptide comprises, in order fromN-terminus to C-terminus: i) the heterologous polypeptide; and ii) theHCV E1 polypeptide. In some cases, the variant HCV E1 polypeptidecomprises, in order from N-terminus to C-terminus: i) the HCV E1polypeptide; and ii) the heterologous polypeptide. In some cases, thevariant HCV E1 polypeptide comprises an immunoglobulin Fc polypeptide.In some cases, the variant HCV E1 polypeptide comprises, in order fromN-terminus to C-terminus: i) the Fc polypeptide; ii) the heterologouspolypeptide; and iii) the HCV E1 polypeptide. In some cases, the variantHCV E1 polypeptide comprises a proteolytic cleavage site interposedbetween the Fc polypeptide and the heterologous polypeptide. In somecases, the proteolytic cleavage site comprises the sequence LEVLFQGP(SEQ ID NO:5), wherein cleavage occurs between the glutamine and theglycine.

The present disclosure nucleic acid comprising a nucleotide sequenceencoding a variant HCV E2 polypeptide as described above or elsewhereherein, or a variant HCV E1 polypeptide as described above or elsewhereherein. The present disclosure provides a recombinant expression vectorcomprising the nucleic acid. In some cases, the nucleotide sequence isoperably linked to a promoter. In some cases, the promoter is functionalin a eukaryotic cell. The present disclosure provides a geneticallymodified in vitro host cell comprising a nucleic acid as described aboveor elsewhere herein, or a recombinant vector as described above orelsewhere herein. In some cases, the host cell is a eukaryotic cell. Insome cases, the host cell is a mammalian cell.

The present disclosure provides a method of inducing an immune responsein an individual, the method comprising administering to the individualan effective amount of a heterodimeric polypeptide as described above orelsewhere herein, or a composition as described above or elsewhereherein. The present disclosure provides a method of inducing an immuneresponse in an individual, the method comprising administering to theindividual an effective amount of a nucleic acid as described above orelsewhere herein.

The present disclosure provides a method of inducing an immune responsein an individual, the method comprising administering to the individualan effective amount of a recombinant expression vector as describedabove or elsewhere herein. In some cases, the recombinant expressionvector is a recombinant modified vaccinia Ankara vector. In some cases,the recombinant expression vector is a recombinant replication-defectiveadenovirus. In some cases, administration of the recombinant expressionvector is by intramuscular administration. In some cases, administrationof the recombinant expression vector is by subcutaneous administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C provide an amino acid sequence alignment of examples of thecore-E1-E2 coding regions of a HCV genotype 1 virus, specificallyrepresentative HCV 1A, 1B and 1C genotypes. Genbank database sequencesfor the coding region core-E1-E2 were aligned using Geneious softwarev5.6.4. Numbering of amino acids is according to strain NP_671941 (H77).Consensus: SEQ ID NO:74; AVI1a129: SEQ ID NO:75; NP_671491 (H77): SEQ IDNO:76; EU155269: SEQ ID NO:77; EU781810: SEQ ID NO:78; EU781771: SEQ IDNO:79; AB250610: SEQ ID NO:80; EU781752: SEQ ID NO:81; EU781759: SEQ IDNO:82; EF407439: SEQ ID NO:83; EF407427: SEQ ID NO:84; EU362905: SEQ IDNO:85; EF407413: SEQ ID NO:86; EU781808: SEQ ID NO:87; EU78170: SEQ IDNO:88; AJ238799 (Con1): SEQ ID NO:89; AAK97744: SEQ ID NO:90; AF139594:SEQ ID NO:91; AF176573: SEQ ID NO:92; BAA19625: SEQ ID NO:93; BAA25076:SEQ ID NO:94; BAC54896: SEQ ID NO:95; BAD91386: SEQ ID NO:96; BAF46764:SEQ ID NO:97; BAG30950: SEQ ID NO:98; CAB41951: SEQ ID NO:99; AAK95832:SEQ ID NO:100; AAT69968: SEQ ID NO:101; and BAA03581: SEQ ID NO:102.

FIG. 2A-2C provide an alignment of amino acid sequences of thecore-E1-E2 coding region of representative HCV 2A and HCV2B subtypes.Genbank database sequences for the coding region core-E1-E2 were alignedusing Geneious software v5.6.4. The amino acid numbering depicted is inaccordance to the common HCV strains: AB047639 (JFH1) and HPCJ8G-J8 (J8)for HCV2A and HCV2B, respectively. AB047639 (JFH1): SEQ ID NO:103;AB047645: SEQ ID NO:104; AF169003: SEQ ID NO:105; AF169005: SEQ IDNO:106; AF238482: SEQ ID NO:107; AY746460: SEQ ID NO:108; HPCPOLP: SEQID NO:109; NC_009823: SEQ ID NO:110; HPCJ8G HC-J8: SEQ ID NO:111;AB030907: SEQ ID NO:112; AY232730: SEQ ID NO:113; AY232747: SEQ IDNO:114; and DQ430817: SEQ ID NO:115.

FIG. 3A-3C provide an amino acid sequence alignment of the core-E1-E2coding region for representative HCV 3A, 3B and 3K genotypes. Genbankdatabase sequences for the coding region core-E1-E2 were aligned usingGeneious software v5.6.4. Consensus: SEQ ID NO:116; AVI3a177: SEQ IDNO:117; ADF97232(S52): SEQ ID NO:118; YP_0014696: SEQ ID NO:119;CAA54244: SEQ ID NO:120; AAC03058: SEQ ID NO:121; AAY29642: SEQ IDNO:122; ABD85062: SEQ ID NO:123; ABD85063: SEQ ID NO:124; ABD97104: SEQID NO:125; BAA06044: SEQ ID NO:126; BAA08372: SEQ ID NO:127; andBAA09890: SEQ ID NO:128.

FIG. 4A-4B provide an amino acid sequence of the core-E1-E2 codingregion for HCV genotype 7a. Amino acid sequence for the coding regioncore-E1-E2 of genotype 7a (isolate QC69; Genbank: ABN05226.1; SEQ IDNO:129) is shown according to the numbering scheme of the referencestrain, NP_671941 (H77).

FIG. 5A-5B present schematic representations of Fc-tagged and untaggedE1E2 expression constructs and polypeptide processing. FIG. 5A depicts aschematic representation of an Fc-tagged E1E2 expression construct andpolypeptide processing. SP denotes signal peptidase cleavage site. PPdenotes cleavage site for precision protease. TPx denotes the additionof a polytope (a polypeptide comprising T-cell epitope(s)) to the E1E2polypeptide at the N-terminus of E2. SEQ ID NO:5. FIG. 5B depicts aschematic representation of an un-tagged E1E2 expression construct andpolypeptide processing.

FIG. 6A-6B depict an alignment of Fc-tagged E1-E2 polypeptide, with andwithout a polytope (TPx) for H77 (GenBank NP_671941) and Alberta isolateAvi1a129 (genotype 1A). AVI1a129: SEQ ID NO:130; AVI1a129TP29: SEQ IDNO:131; AVI1a29TP52: SEQ ID NO:132; AVI1a129TP100: SEQ ID NO:133; H77:SEQ ID NO:134; H77 TP29: SEQ ID NO:135; H77 TP52: SEQ ID NO:136; H77TP100: SEQ ID NO:137.

FIG. 7A-7B depict an alignment of Fc-tagged E1-E2 polypeptide, with andwithout a polytope (TPx) for S52 (GenBank ADF97232.1) and Albertaisolate Avi3a177 (genotype 3A). S52: SEQ ID NO:138; S52 TP29: SEQ IDNO:139; S52 TP52: SEQ ID NO:140; S52 TP100: SEQ ID NO:141; AVI3a177: SEQID NO:142; AVI3a177 TP29: SEQ ID NO:143; AVI3a177 TP52: SEQ ID NO:144;AVI3a177 TP100: SEQ ID NO:145.

FIG. 8A-8B depict purification of an E1E2 heterodimer from CHO cellextracts expressing Fc-tagged E1E2.

FIG. 9A-9C provide amino acid sequences of immunoglobulin Fc regions(SEQ ID NOs:146-153).

FIG. 10 presents Table 1, which provides conserved regions based onconserved CD4 epitopes (CD4⁺ T cell epitopes) (SEQ ID NOs:154-164).

FIG. 11 presents Table 2, which provides the number of located HCV CD8⁺T cell epitopes and anchor positions for common human leukocyte antigen(HLA)-I Alleles in the United States.

FIG. 12 presents Table 3, which provides conserved regions based onconserved CD8 epitopes (CD8⁺ T cell epitopes) (SEQ ID NOs:165-174).

FIG. 13A-13B provide a list of CD4 and CD8 epitopes that are conservedamond HCV genotypes 1a, 1b, 2a, 2b, and 3.

FIG. 14A-14D provide amino acid sequences of examples of T-cellpolytopes (“TP”). The start and end amino acids are based on thesequence designated “Consensus” in FIG. 16A-16L. The T-cell epitopescontained within each TP are provided; the T-cell epitope designationscorrespond to those presented in FIG. 15A-15N (SEQ ID NOs:175-184).

FIG. 15A-15N provide consensus amino acid sequences of HCV polypeptides;and depict the locations of T-cell epitopes (SEQ ID NO:185).

FIG. 16A-16L provide consensus amino acid sequences of HCV polypeptides(SEQ ID NOs:186-197).

DEFINITIONS

The term “hepatitis C virus” (“HCV”), as used herein, refers to any oneof a number of different genotypes and isolates of hepatitis C virus.Thus, “HCV” encompasses any of a number of genotypes, subtypes, orquasispecies, of HCV, including, e.g., genotype 1, 2, 3, 4, 6, 7, etc.and subtypes (e.g., 1a, 1b, 2a, 2b, 3a, 4a, 4c, etc.), and quasispecies.Representative HCV genotypes and isolates include: the “Chiron” isolateHCV-1, H77, J6, Con1, isolate 1, BK, EC1, EC10, HC-J2, HC-J5; HC-J6,HC-J7, HC-J8, HC-JT, HCT18, HCT27, HCV-476, HCV-KF, “Hunan”, “Japanese”,“Taiwan”, TH, type 1, type 1a, H77 type 1b, type 1c, type 1d, type 1e,type 1f, type 10, type 2, type 2a, type 2b, type 2c, type 2d, type 2f,type 3, type 3a, type 3b, type 3g, type 4, type 4a, type 4c, type 4d,type 4f, type 4h, type 4k, type 5, type 5a, type 6 and type 6a.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, non-human primates (e.g., simians), equines (e.g., horses),and humans.

As used herein, the term “isolated,” in reference to a polypeptide,refers to a polypeptide that is in an environment different from that inwhich the polypeptide naturally occurs. An isolated polypeptide can bepurified. By “purified” is meant a compound of interest (e.g., apolypeptide) has been separated from components that accompany it innature. “Purified” can also be used to refer to a polypeptide separatedfrom components that can accompany it during production of thepolypeptide (e.g., during synthesis in vitro, etc.). In someembodiments, a polypeptide (or a mixture of polypeptides) issubstantially pure when the polypeptide (or mixture of polypeptides) isat least 60% or at least 75% by weight free from organic molecules withwhich it is naturally associated or with which it is associated duringproduction. In some embodiments, the polypeptide is from 30% to 60%pure. In some embodiments, the polypeptide (or mixture of polypeptides)is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, or at least 99%, by weight, pure. For example, in someembodiments, an E1 or an E2 polypeptide (or a mixture of E1 and E2polypeptides) is substantially pure when the E1 or E2 polypeptide (ormixture of E1 and E2 polypeptides) is at least 60% or at least 75% byweight free from organic molecules with which the polypeptide(s) isnaturally associated or with which it is associated during production.In some embodiments, the E1 or E2 polypeptide (or mixture of E1 and E2polypeptides) is at least 60%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 99%, by weight, pure. In someembodiments, where a composition comprises an E2 polypeptide, the E2polypeptide is at least 60%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 99%, by weight, pure. In someembodiments, where a composition comprises an E1/E2 heterodimericcomplex polypeptide, the E1/E2 heterodimeric complex polypeptide is atleast 60%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99%, by weight, pure. In some embodiments, wherea composition comprises an E1/variant E2 heterodimeric complexpolypeptide, the E1/variant E2 heterodimeric complex polypeptides are atleast 60%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99%, by weight, pure. In some embodiments, wherea composition comprises a variant E1/E2 heterodimeric complexpolypeptide, the variant E1/E2 heterodimeric complex polypeptides are atleast 60%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99%, by weight, pure. In some embodiments, wherea composition comprises a variant E1/variant E2 heterodimeric complexpolypeptide, the variant E1/variant E2 heterodimeric complexpolypeptides are at least 60%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 99%, by weight, pure. In someembodiments, where a composition comprises a variant E1 polypeptide, thevariant E1 polypeptide is at least 60%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99%, by weight, pure.In some embodiments, where a composition comprises a variant E2polypeptide, the variant E2 polypeptide is at least 60%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%,by weight, pure.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases. In some cases, apolynucleotide is RNA. In some cases, a polynucleotide is DNA. A“polynucleotide” includes a nucleic acid that is incorporated into aviral vector or a bacterial vector.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term “polypeptide”includes glycosylated polypeptides.

The term “heterologous” refers to two components that are defined bystructures derived from different sources. For example, where“heterologous” is used in the context of a polypeptide, where thepolypeptide includes operably linked amino acid sequences that can bederived from one or more different polypeptides, e.g., amino acidsequences that are not operably linked to the polypeptide in nature.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “avariant HCV E2 polypeptide” includes a plurality of such polypeptidesand reference to “the HCV E1 polypeptide” includes reference to one ormore HCV E1 polypeptides and equivalents thereof known to those skilledin the art, and so forth. It is further noted that the claims may bedrafted to exclude any optional element. As such, this statement isintended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

T cell responses and antibody responses to HCV can be protective againstHCV infection. The present disclosure provides heterodimericpolypeptides comprising: 1) a variant hepatitis C virus (HCV) E2polypeptide and an HCV E1 polypeptide; 2) a variant HCV E1 polypeptideand an HCV E2 polypeptide; and 3) a variant HCV E1 polypeptide and avariant HCV E2 polypeptide, where the variant HCV E2 polypeptide and/oror the HCV E1 polypeptide comprises a heterologous polypeptidecomprising one or more T cell epitopes, e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide, where the one or more T-cell epitopes arereferred to as a “polytope.” Inclusion of T cell epitopes provides for amore robust T-cell response to HCV, including cytotoxic CD8⁺ T-cellresponses to HCV-infected cells, and CD4⁺ T helper responses. EnhancedCD4⁺ responses may also result in a higher titer of neutralizinganti-E1/E2 antibodies to HCV. Such T-cell responses and antibody titerscan provide a protective response against HCV infection. The T-cellepitopes that are included within the polytope can include conservedT-cell epitopes and/or immunodominant T-cell epitopes. It was found thatinclusion, in an HCV E2 polypeptide, of a heterologous polypeptidecomprising a polytope (one or more T-cell epitopes) allows formation ofan E1/E2 heterodimer. A purification scheme was devised, which providesfor ease of production of E1/E2 heterodimers (where one or both of theE1 and E2 polypeptides comprises a heterologous polypeptide comprisingone or more T-cell epitopes), and which provides for highly purifiedE1/E2 heterodimers using a scaleable vaccine manufacturing levelprocess.

E1/E2 heterodimers of the present disclosure provide improvements topreviously-described vaccine candidates by 1) eliciting higher levels ofHCV-specific CD4⁺ T helper responses, which are known to contribute toprotection against HCV infection; 2) eliciting higher levels ofHCV-specific CD8⁺ cytotoxic T cell responses, which are known tocontribute to protection against HCV infection; and 3) via the inclusionof extra CD4⁺ T helper epitopes, leading to higher titers of HCVneutralizing antibodies which are also known to be associated withprotection against HCV infection.

A variant E2 polypeptide, also referred to as an “E2 fusionpolypeptide,” comprises an HCV E2 polypeptide and a heterologouspolypeptide, where the heterologous polypeptide is also referred to as a“fusion partner.” The heterologous polypeptide is covalently linked tothe HCV E2 polypeptide. A variant E1 polypeptide, also referred to as an“E1 fusion polypeptide,” comprises an HCV E1 polypeptide and aheterologous polypeptide, where the heterologous polypeptide is alsoreferred to as a “fusion partner.” The heterologous polypeptide iscovalently linked to the HCV E1 polypeptide. The heterologouspolypeptide (“fusion partner”) comprises one or more T-cell epitopespresent in an HCV polypeptide other than an HCV E1 polypeptide or an HCVE2 polypeptide. The heterologous polypeptide can be fused to theN-terminus or the C-terminus of the HCV E1 or HCV E2 polypeptide.

The present disclosure provides heterodimeric polypeptides comprising avariant HCV E2 polypeptide and an HCV E1 polypeptide, or comprising avariant HCV E1 polypeptide and an HCV E2 polypeptide, or comprising avariant HCV E1 polypeptide and a variant HCV E2 polypeptide, where thevariant HCV E2 polypeptide and/or the HCV E1 polypeptide comprises aheterologous polypeptide comprising one or more T cell epitopes, e.g.,one or more T cell epitopes present in an HCV polypeptide other than anHCV E1 polypeptide or an HCV E2 polypeptide. The present disclosureprovides nucleic acids encoding a polyprotein that includes E1 andvariant E2, or that includes E2 and variant E1, or that includes variantE2 and variant E1. The present disclosure provides a method of producingan E1/E2 heterodimer of the present disclosure. The present disclosurealso provides variant E2 polypeptides and variant E1 polypeptides; andnucleic acids encoding the variant polypeptides. The present disclosureprovides a method of inducing an immune response in an individual. Thepresent disclosure provides a method of inducing an immune response(e.g., a protective immune response) to HCV antigens in an individual.The present disclosure provides a method of inducing a protective immuneresponse to one or more HCV genotypes in an individual. In some cases,the HCV E2 polypeptide is an HCV E2 ectodomain polypeptide. In somecases, the HCV E2 polypeptide is a full-length HCV E2 polypeptide. Insome cases, the HCV E1 polypeptide is an HCV E1 ectodomain polypeptide.In some cases, the HCV E1 polypeptide is a full-length HCV E1polypeptide.

The present disclosure provides heterodimeric polypeptides comprising:a) a variant HCV E2 polypeptide and an HCV E1 polypeptide; b) a variantHCV E1 polypeptide and an HCV E2 polypeptide; or c) a variant HCV E1polypeptide and a variant HCV E2 polypeptide, where the variant HCV E2polypeptide or the HCV E1 polypeptide comprises one or more T cellepitopes, e.g., one or more T cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide.The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV non-structural polypeptide-3 (NS3) polypeptide; b)an HCV non-structural polypeptide-2 (NS2) polypeptide; c) an HCVnon-structural polypeptide-4A (NS4A) polypeptide; d) an HCVnon-structural polypeptide-4B (NS4B) polypeptide; e) an HCVnon-structural polypeptide-5A (NS5A) polypeptide; f) an HCVnon-structural polypeptide-5B (NS5B) polypeptide; g) an HCV corepolypeptide; or h) an HCV p7 polypeptide. In some cases, the one or moreT-cell epitopes are T-cell epitopes present in an HCV NS3 polypeptide.In some cases, the heterologous polypeptide further comprises one ormore T cell epitopes present in: a) cholera toxin or toxoid; and/or b)tetanus toxin or toxoid; and/or c) diphtheria toxin or toxoid; and/or d)a meningococcal outer membrane protein. Thus, in some cases, a variantHCV E1 polypeptide or variant HCV E2 polypeptide of an E1/E2 heterodimerof the present disclosure includes: a) an HCV E1 polypeptide or an HCVE2 polypeptide; and b) a heterologous polypeptide that comprises one ormore T-cell epitopes, where the one or more T-cell epitopes are T-cellepitopes present in: i) one or more of an HCV NS3 polypeptide, an HCVNS2 polypeptide, an HCV NS4A polypeptide, an HCV NS4B polypeptide, anHCV NS5A polypeptide, an HCV NS5B polypeptide, an HCV core polypeptide,and an HCV p7 polypeptide; and ii) one or more of cholera toxin ortoxoid, tetanus toxin or toxoid, diphtheria toxin or toxoid, and ameningococcal outer membrane protein.

In some cases, a heterodimeric polypeptide of the present disclosureincludes: a) an HCV E1 polypeptide; and b) a variant HCV E2 polypeptidecomprising: i) an HCV E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide). The one or more T-cell epitopes can includeone or more T-cell epitopes present in: a) an HCV NS3 polypeptide; b) anHCV NS2 polypeptide; c) an HCV NS4A polypeptide; d) an HCV NS4Bpolypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5B polypeptide; g)an HCV core polypeptide; or h) an HCV p7 polypeptide. In some cases, theone or more T-cell epitopes are T-cell epitopes present in an HCV NS3polypeptide. In some cases, the heterologous polypeptide furthercomprises one or more T cell epitopes present in: a) cholera toxin ortoxoid; and/or b) tetanus toxin or toxoid; and/or c) diphtheria toxin ortoxoid; and/or d) a meningococcal outer membrane protein. In some cases,a variant HCV E2 polypeptide of an E1/E2 heterodimer of the presentdisclosure includes: a) an HCV E2 polypeptide; and b) a heterologouspolypeptide that comprises one or more T-cell epitopes, where the one ormore T-cell epitopes are T-cell epitopes present in: i) one or more ofan HCV NS3 polypeptide, an HCV NS2 polypeptide, an HCV NS4A polypeptide,an HCV NS4B polypeptide, an HCV NS5A polypeptide, an HCV NS5Bpolypeptide, an HCV core polypeptide, and an HCV p7 polypeptide; and ii)one or more of cholera toxin or toxoid, tetanus toxin or toxoid,diphtheria toxin or toxoid, and a meningococcal outer membrane protein

In some cases, a heterodimeric polypeptide of the present disclosureincludes: a) a variant HCV E1 polypeptide comprising: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide); and b) an HCV E2 polypeptide. The one or more T-cellepitopes can include one or more T-cell epitopes present in: a) an HCVNS3 polypeptide; b) an HCV NS2 polypeptide; c) an HCV NS4A polypeptide;d) an HCV NS4B polypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5Bpolypeptide; g) an HCV core polypeptide; or h) an HCV p7 polypeptide. Insome cases, the one or more T-cell epitopes are T-cell epitopes presentin an HCV NS3 polypeptide. In some cases, the heterologous polypeptidefurther comprises one or more T cell epitopes present in: a) choleratoxin or toxoid; and/or b) tetanus toxin or toxoid; and/or c) diphtheriatoxin or toxoid; and/or d) a meningococcal outer membrane protein. Insome cases, a variant HCV E1 polypeptide of an E1/E2 heterodimer of thepresent disclosure includes: a) an HCV E1 polypeptide; and b) aheterologous polypeptide that comprises one or more T-cell epitopes,where the one or more T-cell epitopes are T-cell epitopes present in: i)one or more of an HCV NS3 polypeptide, an HCV NS2 polypeptide, an HCVNS4A polypeptide, an HCV NS4B polypeptide, an HCV NS5A polypeptide, anHCV NS5B polypeptide, an HCV core polypeptide, and an HCV p7polypeptide; and ii) one or more of cholera toxin or toxoid, tetanustoxin or toxoid, diphtheria toxin or toxoid, and a meningococcal outermembrane protein.

In some cases, a heterodimeric polypeptide of the present disclosureincludes: a) a variant HCV E1 polypeptide comprising: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide); and b) a variant HCV E2 polypeptide comprising: i) an HCVE2 polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). The one or more T-cell epitopes can include one or moreT-cell epitopes present in: a) an HCV NS3 polypeptide; b) an HCV NS2polypeptide; c) an HCV NS4A polypeptide; d) an HCV NS4B polypeptide; e)an HCV NS5A polypeptide; f) an HCV NS5B polypeptide; g) an HCV corepolypeptide; or h) an HCV p7 polypeptide. In some cases, the one or moreT-cell epitopes are T-cell epitopes present in an HCV NS3 polypeptide.In some cases, the heterologous polypeptide further comprises one ormore T cell epitopes present in: a) cholera toxin or toxoid; and/or b)tetanus toxin or toxoid; and/or c) diphtheria toxin or toxoid; and/or d)a meningococcal outer membrane protein. In some cases, a variant HCV E2and a variant HCV E1 polypeptide of an E1/E2 heterodimer of the presentdisclosure includes: 1) a) an HCV E2 polypeptide; and b) a heterologouspolypeptide that comprises one or more T-cell epitopes, where the one ormore T-cell epitopes are T-cell epitopes present in: i) one or more ofan HCV NS3 polypeptide, an HCV NS2 polypeptide, an HCV NS4A polypeptide,an HCV NS4B polypeptide, an HCV NS5A polypeptide, an HCV NS5Bpolypeptide, an HCV core polypeptide, and an HCV p7 polypeptide; and ii)one or more of cholera toxin or toxoid, tetanus toxin or toxoid,diphtheria toxin or toxoid, and a meningococcal outer membrane protein;and 2) a) an HCV E1 polypeptide; and b) a heterologous polypeptide thatcomprises one or more T-cell epitopes, where the one or more T-cellepitopes are T-cell epitopes present in: i) one or more of an HCV NS3polypeptide, an HCV NS2 polypeptide, an HCV NS4A polypeptide, an HCVNS4B polypeptide, an HCV NS5A polypeptide, an HCV NS5B polypeptide, anHCV core polypeptide, and an HCV p7 polypeptide; and ii) one or more ofcholera toxin or toxoid, tetanus toxin or toxoid, diphtheria toxin ortoxoid, and a meningococcal outer membrane protein.

As noted above, the present disclosure provides heterodimericpolypeptides comprising: a) a variant HCV E2 polypeptide and an HCV E1polypeptide; b) a variant HCV E1 polypeptide and an HCV E2 polypeptide;or c) a variant HCV E1 polypeptide and a variant HCV E2 polypeptide,where the variant HCV E2 polypeptide or the HCV E1 polypeptide comprisesone or more T cell epitopes, e.g., one or more T cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide. The presence of the one or more T-cell epitopes (e.g., oneor more T-cell epitopes conserved among the hepacivirus genus; e.g., oneor more immunodominant T-cell epitopes) provides for a more robustcellular immune response (e.g., a CD4⁺ and/or a CD8⁺ immune response) toHCV than a wild-type HCV E1/E2 heterodimer. For example, the addition ofthe one or more T-cell epitopes provides for a more robust CD4⁺ helperand CD8⁺ cytotoxic T cell response to HCV than a wild-type HCV E1/E2heterodimer, and provides greater T helper activity to promote strongerantibody responses to the E1/E2 heterodimer. These features provide forsuperior HCV vaccine antigens.

The present disclosure provides variant HCV E2 polypeptides, and variantHCV E1 polypeptides. A variant E2 polypeptide of the present disclosureheterodimerizes with an HCV E1 polypeptide. A variant E1 polypeptide ofthe present disclosure heterodimerizes with an HCV E2 polypeptide. Theheterodimer, or a polynucleotide(s) comprising a nucleotide sequenceencoding the heterodimer, can be used to induce an immune responseagainst HCV in an individual. For example, the heterodimer, or apolynucleotide(s) comprising a nucleotide sequence encoding theheterodimer, can be used to induce a protective immune response againstHCV in an individual. In some cases, the heterodimer, or apolynucleotide(s) comprising a nucleotide sequence encoding theheterodimer, can be used to induce a protective immune response againstHCV of more than one genotype.

Suitable T-cell epitopes (e.g., one or more conserved T-cell epitopes;e.g., one or more immunodominant T-cell epitopes) are described indetail below; and in the Figures. Suitable T-cell epitopes can beidentified using the methods described in the Examples section, or usingany other method that identifies conserved T-cell epitopes orimmunodominant T-cell epitopes.

In some cases, a heterologous polypeptide present in a variant E1polypeptide or a variant E2 polypeptide described herein, where theheterologous polypeptide comprises one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide, comprises: a) two or more (2, 3, 4, 5, 6, 7, 8, 9, or 10,or more than 10) T cell epitopes of an HCV NS3 polypeptide; b) two ormore (2, 3, 4, 5, 6, 7, 8, 9, or 10, or more than 10) T cell epitopes ofan HCV NS2 polypeptide; c) two or more (2, 3, 4, 5, 6, 7, 8, 9, or 10,or more than 10) T cell epitopes of an HCV NS4A polypeptide; d) two ormore (2, 3, 4, 5, 6, 7, 8, 9, or 10, or more than 10) T cell epitopes ofan HCV NS4B polypeptide; e) two or more (2, 3, 4, 5, 6, 7, 8, 9, or 10,or more than 10) T cell epitopes of an HCV NS5A polypeptide; f) two ormore (2, 3, 4, 5, 6, 7, 8, 9, or 10, or more than 10) T cell epitopes ofan HCV NS5B polypeptide; g) two or more (2, 3, 4, 5, 6, 7, 8, 9, or 10,or more than 10) T cell epitopes of an HCV core polypeptide; h) two ormore (2, 3, 4, 5, 6, 7, 8, 9, or 10, or more than 10) T cell epitopes ofan HCV p7 polypeptide; i) one or more T-cell epitopes of an HCV NS3polypeptide and one or more T-cell epitopes of an HCV NS2 polypeptide;j) one or more T-cell epitopes of an HCV NS3 polypeptide and one or moreT-cell epitopes of an HCV NS4B polypeptide; k) one or more T-cellepitopes of an HCV NS3 polypeptide and one or more T-cell epitopes of anHCV NS5A polypeptide; l) one or more T-cell epitopes of an HCV NS3polypeptide and one or more T-cell epitopes of an HCV NSSB polypeptide;m) one or more T-cell epitopes of an HCV NS3 polypeptide and one or moreT-cell epitopes of an HCV core polypeptide; n) one or more T-cellepitopes of an HCV NS3 polypeptide, one or more T-cell epitopes of anHCV NS2 polypeptide, and one or more T-cell epitopes of an HCV corepolypeptide; o) one or more T-cell epitopes of an HCV NS3 polypeptide,one or more T-cell epitopes of an HCV NS2 polypeptide, and one or moreT-cell epitopes of an HCV NS4B polypeptide; or p) one or more T-cellepitopes of an HCV NS3 polypeptide, one or more T-cell epitopes of anHCV NS2 polypeptide, and one or more T-cell epitopes of an HCV NSSApolypeptide. Other combinations are possible and are contemplated by thepresent disclosure.

In some cases, a variant E2 polypeptide and/or a variant E1 polypeptidecomprises a heterologous polypeptide comprising a polytope thatcomprises: 1) a contiguous NS3-NS4a polypeptide in which the NS3-encodedserine protease is rendered inactive by mutation of any one of thecatalytic triad amino acids (H,D,S); 2) a contiguous NS3-NS4a-NS4apolypeptide in which the NS3-encoded serine protease is renderedinactive by mutation of any one of the catalytic triad amino acids(H,D,S); 3) a contiguous NS3-NS4a-NS4a-NS5a polypeptide in which theNS3-encoded serine protease is rendered inactive by mutation of any oneof the catalytic triad amino acids (H,D,S); or 4) a contiguousNS3-NS4a-NS4a-NS5a-NS5a polypeptide in which the NS3-encoded serineprotease is rendered inactive by mutation of any one of the catalytictriad amino acids (H,D,S) and the NS5b-encoded RNA polymerase isrendered inactive by mutation of any residues in the GDD motif that isrequired for polymerase activity.

In some cases, a linker can be interposed between the heterologouspolypeptide (“polytope”) and the HCV E1 or HCV E2 polypeptide. Thelinker peptide may have any of a variety of amino acid sequences. Alinker can be a peptide of between about 6 and about 40 amino acids inlength, or between about 6 and about 25 amino acids in length. Theselinkers can be produced by using synthetic, linker-encodingoligonucleotides to couple the proteins. Peptide linkers allowing adegree of flexibility can be used. The linking peptides may havevirtually any amino acid sequence, bearing in mind that suitable linkerswill have a sequence that results in a generally flexible peptide. Theuse of small amino acids, such as glycine and alanine, are of use increating a flexible peptide. The creation of such sequences is routineto those of skill in the art.

Suitable linkers can be readily selected and can be of any of a suitableof different lengths, such as from 1 amino acid (e.g., Gly, Ala, or Ser)to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 aminoacids to 12 amino acids, including 4 amino acids to 10 amino acids, 5amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 aminoacids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), (GSGGS)_(n)(SEQ ID NO:55) and (GGGS)_(n) (SEQ ID NO:56), where n is an integer ofat least one), glycine-alanine polymers, alanine-serine polymers, andother flexible linkers known in the art. Glycine and glycine-serinepolymers are of interest since both of these amino acids are relativelyunstructured, and therefore may serve as a neutral tether betweencomponents. Glycine polymers are of particular interest since glycineaccesses significantly more phi-psi space than even alanine, and is muchless restricted than residues with longer side chains (see Scheraga,Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkersinclude, but are not limited to, GGSG (SEQ ID NO:57), GGSGG (SEQ IDNO:58), GSGSG (SEQ ID NO:59), GSGGG (SEQ ID NO:60), GGGSG (SEQ IDNO:61), GSSSG (SEQ ID NO:62), and the like. The ordinarily skilledartisan will recognize that design of a peptide conjugated to anyelements described above can include linkers that are all or partiallyflexible, such that the linker can include a flexible linker as well asone or more portions that confer less flexible structure.

I. E1/E2 Heterodimers

The present disclosure provides heterodimeric polypeptides comprising:a) a variant HCV E2 polypeptide and an HCV E1 polypeptide; b) a variantHCV E1 polypeptide and an HCV E2 polypeptide; or c) a variant HCV E1polypeptide and a variant HCV E2 polypeptide, where the variant HCV E2polypeptide or the HCV E1 polypeptide comprises one or more T cellepitopes, e.g., one or more T cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide.

An E1/E2 heterodimer of the present disclosure, or a nucleic acid(s)(e.g., one or more recombinant expression vectors; an mRNA molecule(s);a DNA molecule(s)) comprising a nucleotide sequence encoding the E1/E2heterodimer, can be used to induce an immune response to HCV in anindividual. The nucleic acid may be in the form of DNA or RNA, or may becomplexed with a polymer such as poly-lactic-co-glycolide (PLG) orliposomal formulations or may be inserted into a viral vaccine vector orbacterial vaccine vectors. Nucleic acid vaccines are feasible usingeither viral or bacterial vectors to deliver the nucleic acids encodingvaccine antigens or by delivering the encoding DNA or RNA into animmunogenic vaccine formulation (Deering et al. (2014) Expert Opin DrugDeliv. 11(6):885-99).

IA. E1E2 Heterodimers Comprising HCV E1 and a Variant HCV E2

The present disclosure provides an E1/E2 heterodimer, where the E1/E2heterodimer comprises: a) a variant HCV E2 polypeptide, where thevariant HCV E2 polypeptide comprises: i) an HCV E2 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T-cell epitopes not present in an HCV E1 or an HCV E2polypeptide; e.g., one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 or an HCV E2 polypeptide); and b) anHCV E1 polypeptide. Thus, in some cases, a heterodimeric polypeptide ofthe present disclosure includes: a) an HCV E1 polypeptide; and b) avariant HCV E2 polypeptide comprising: i) an HCV E2 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). The one or moreT-cell epitopes can include one or more T-cell epitopes present in: a)an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) an HCV NS4Apolypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5A polypeptide; f)an HCV NS5B polypeptide; g) an HCV core polypeptide; or h) an HCV p7polypeptide. In some cases, the one or more T-cell epitopes are T-cellepitopes present in an HCV NS3 polypeptide. In some cases, theheterologous polypeptide further comprises one or more T cell epitopespresent in: a) cholera toxin or toxoid; and/or b) tetanus toxin ortoxoid; and/or c) diphtheria toxin or toxoid; and/or d) a meningococcalouter membrane protein. The heterologous polypeptide is also referred toas a “polytope.”

An E1/E2 heterodimer of the present disclosure (e.g., an E1/E2heterodimer comprising: a) an HCV E1 polypeptide; and b) a variant HCVE2 polypeptide comprising: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes), whenadministered to an individual in need thereof, induces an immuneresponse in the individual to one or more HCV genotypes. An E1/E2heterodimer of the present disclosure, when administered to anindividual in need thereof, induces an immune response in the individualto one or more HCV genotypes, where the immune response is greater thanthe immune response induced by administration of an HCV E1/E2heterodimer comprising a wild-type E1 and a wild-type E2 polypeptide oran E2 polypeptide lacking the polytope.

For example, in some cases, an E1/E2 heterodimer of the presentdisclosure (e.g., an E1/E2 heterodimer comprising: a) an HCV E1polypeptide; and b) a variant HCV E2 polypeptide comprising: i) an HCVE2 polypeptide; and ii) a heterologous polypeptide comprising one ormore T-cell epitopes), when administered to an individual in needthereof, induces cytotoxic T lymphocytes (CTLs) specific for HCV, wherethe number of HCV-specific CTLs induced is at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 7.5-fold, at least 10-fold, at least 20-fold, at least50-fold, or at least 100-fold, or more than 100-fold, higher than thenumber of HCV-specific CTLs induced by administration of an HCV E1/E2heterodimer comprising a wild-type E1 and a wild-type E2 polypeptide oran E2 polypeptide lacking the polytope.

In some cases, an E1/E2 heterodimer of the present disclosure (e.g., anE1/E2 heterodimer comprising: a) an HCV E1 polypeptide; and b) a variantHCV E2 polypeptide comprising: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces production of HCV-specific CD4⁺ T cells and CD8⁺ T cells in theindividual, where the number of HCV-specific CD4⁺ T cells and CD8⁺ Tcells is increased, such that the percent of the total peripheral bloodT cells (i.e., the total number of CD4⁺ T cells+CD8⁺ T cells in theperipheral blood) that are HCV-specific CD4⁺ T cells and CD8⁺ T cells isfrom 0.05% to 10% (e.g., from 0.05% to 0.1%, from 0.1% to 0.5%, from0.5% to 1%, from 1% to 2%, from 2% to 5%, or from 5% to 10%). The numberof HCV-specific CD4⁺ T cells and CD8⁺ T cells in a control individual(e.g., an individual not infected with HCV) not treated with the E1/E2heterodimer would be undetectable.

For example, in some cases, an E1/E2 heterodimer of the presentdisclosure (e.g., an E1/E2 heterodimer comprising: a) an HCV E1polypeptide; and b) a variant HCV E2 polypeptide comprising: i) an HCVE2 polypeptide; and ii) a heterologous polypeptide comprising one ormore HCV NS3 T-cell epitopes), when administered to an individual inneed thereof, induces production of HCV NS3-specific CD4⁺ T cells andCD8⁺ T cells in the individual, where the number of HCV NS3-specificCD4⁺ T cells and CD8⁺ T cells is increased, such that the percent of thetotal peripheral blood T cells (i.e., the total number of CD4⁺ Tcells+CD8⁺ T cells in the peripheral blood) that are HCV NS3-specificCD4⁺ T cells and CD8⁺ T cells is from 0.05% to 10% (e.g., from 0.05% to0.1%, from 0.1% to 0.5%, from 0.5% to 1%, from 1% to 2%, from 2% to 5%,or from 5% to 10%). The number of HCV NS3-specific CD4⁺ T cells and CD8⁺T cells in a control individual (e.g., an individual not infected withHCV) not treated with the E1/E2 heterodimer would be undetectable.

In some cases, an E1/E2 heterodimer of the present disclosure (e.g., anE1/E2 heterodimer comprising: a) an HCV E1 polypeptide; and b) a variantHCV E2 polypeptide comprising: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,increases the number of HCV E1/E2-specific CD4⁺ T cells and CD8⁺ T cellsin the individual by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 40%, at least 50%, at least 75%, at least100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 7.5-fold,at least 10-fold, at least 20-fold, at least 50-fold, or at least100-fold, or more than 100-fold, compared to the number of HCVE1/E2-specific CD4+ T cells and CD8⁺ T cells in the individual inducedby administration of an E1/E2 heterodimer comprising a wild-type E1polypeptide and a wild-type E2 polypeptide, or an E2 polypeptide lackingthe polytope, or compared to the number of HCV E1/E2-specific CD4⁺ Tcells and CD8+ T cells in the individual before administration of theE1/E2 heterodimer of the present disclosure.

As another example, in some cases, an E1/E2 heterodimer of the presentdisclosure (e.g., an E1/E2 heterodimer comprising: a) an HCV E1polypeptide; and b) a variant HCV E2 polypeptide comprising: i) an HCVE2 polypeptide; and ii) a heterologous polypeptide comprising one ormore T-cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide), when administered to anindividual in need thereof, induces helper T lymphocytes (e.g., CD4⁺ Tcells) specific for HCV, where the number of HCV-specific helper T cellsinduced is at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 40%, at least 50%, at least 75%, at least 100% (or2-fold), at least 2.5-fold, at least 5-fold, at least 7.5-fold, at least10-fold, at least 20-fold, at least 50-fold, or at least 100-fold, ormore than 100-fold, higher than the number of HCV-specific helper Tcells induced by administration of an E1/E2 heterodimer comprising awild-type E1 polypeptide and a wild-type E2 polypeptide, or an E2polypeptide lacking the polytope, or compared to the number ofHCV-specific helper T cells in the individual before administration ofthe E1/E2 heterodimer of the present disclosure.

In some cases, an E1/E2 heterodimer of the present disclosure (e.g., anE1/E2 heterodimer comprising: a) an HCV E1 polypeptide; and b) a variantHCV E2 polypeptide comprising: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces antibody specific for HCV (e.g., anti-E1/E2 antibody), where thelevel of HCV-specific antibody induced is at least at high as the levelof HCV-specific antibody induced by administration of an E1/E2heterodimer comprising a wild-type E1 polypeptide and a wild-type E2polypeptide, or an E2 polypeptide lacking the polytope.

In some cases, an E1/E2 heterodimer of the present disclosure (e.g., anE1/E2 heterodimer comprising: a) an HCV E1 polypeptide; and b) a variantHCV E2 polypeptide comprising: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces antibody specific for HCV (e.g., anti-E1/E2 antibody), where thelevel of HCV-specific antibody induced is at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 7.5-fold, at least 10-fold, at least 20-fold, at least50-fold, or at least 100-fold, or more than 100-fold, higher than thelevel of HCV-specific antibody induced by administration of an E1/E2heterodimer comprising a wild-type E1 polypeptide and a wild-type E2polypeptide, or an E2 polypeptide lacking the polytope.

An E1/E2 heterodimer of the present disclosure (e.g., an E1/E2heterodimer comprising: a) an HCV E1 polypeptide; and b) a variant HCVE2 polypeptide comprising: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces an immune response (e.g., a cellular immune response) in theindividual to one or more HCV genotypes. In some cases, an E1/E2heterodimer of the present disclosure (e.g., an E1/E2 heterodimercomprising: a) an HCV E1 polypeptide; and b) a variant HCV E2polypeptide comprising: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide comprising one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide),when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 1. In some cases, an E1/E2heterodimer of the present disclosure (e.g., an E1/E2 heterodimercomprising: a) an HCV E1 polypeptide; and b) a variant HCV E2polypeptide comprising: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide comprising one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide),when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 2. In some cases, an E1/E2heterodimer of the present disclosure (e.g., an E1/E2 heterodimercomprising: a) an HCV E1 polypeptide; and b) a variant HCV E2polypeptide comprising: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide comprising one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide),when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 3. In some cases, an E1/E2heterodimer of the present disclosure (e.g., an E1/E2 heterodimercomprising: a) an HCV E1 polypeptide; and b) a variant HCV E2polypeptide comprising: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide comprising one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide),when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 1 and HCV genotype 3. In somecases, an E1/E2 heterodimer of the present disclosure (e.g., an E1/E2heterodimer comprising: a) an HCV E1 polypeptide; and b) a variant HCVE2 polypeptide comprising: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces an immune response in the individual to HCV genotype 1, HCVgenotype 2, and HCV genotype 3. In some cases, an E1/E2 heterodimer ofthe present disclosure (e.g., an E1/E2 heterodimer comprising: a) an HCVE1 polypeptide; and b) a variant HCV E2 polypeptide comprising: i) anHCV E2 polypeptide; and ii) a heterologous polypeptide comprising one ormore T-cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide), when administered to anindividual in need thereof, induces an immune response in the individualto HCV genotype 1, HCV genotype 2, HCV genotype 3, and HCV genotype 7.In some cases, an E1/E2 heterodimer of the present disclosure (e.g., anE1/E2 heterodimer comprising: a) an HCV E1 polypeptide; and b) a variantHCV E2 polypeptide comprising: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces an immune response in the individual to 1, 2, 3, 4, 5, 6, orall, of HCV genotype 1, HCV genotype 2, HCV genotype 3, HCV genotype 4,HCV genotype 5, HCV genotype 6, and HCV genotype 7. In some cases, anE1/E2 heterodimer of the present disclosure (e.g., an E1/E2 heterodimercomprising: a) an HCV E1 polypeptide; and b) a variant HCV E2polypeptide comprising: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide comprising one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide),when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 1, HCV genotype 2, HCVgenotype 3, HCV genotype 4, HCV genotype 5, HCV genotype 6, and HCVgenotype 7.

Variant E2

As noted above, a variant E2 polypeptide of an HCV E1/E2 heterodimer ofthe present disclosure comprises: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T cell epitopes (e.g.,one or more T-cell epitopes not present in an HCV E1 or an HCV E2polypeptide; e.g., one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 or an HCV E2 polypeptide). In somecases, the variant HCV E2 polypeptide comprises, in order from aminoterminus (N-terminus) to carboxyl terminus (C-terminus): i) an HCV E2polypeptide; and ii) a heterologous polypeptide comprising one or more Tcell epitopes. In some cases, the variant HCV E2 polypeptide comprises,in order from N-terminus to C-terminus: i) a heterologous polypeptidecomprising one or more T cell epitopes; and ii) an HCV E2 polypeptide.

In some cases, a variant E2 polypeptide of an HCV E1/E2 heterodimer ofthe present disclosure comprises from 1 to 10 amino acids at theN-terminus of the variant E2 polypeptide, which 1 to 10 amino acids arepart of a cleavable linker that remains following cleavage of apolyprotein precursor, as described below. For example, where thecleavable linker comprises the amino acid sequence LEVLFQGP (SEQ IDNO:5), the variant E2 polypeptide can comprise Gly-Pro residues at theN-terminus of the polypeptide, e.g., as depicted in FIG. 5A.

E2

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can have a length of fromabout 200 amino acids (aa) to about 250 aa, from about 250 aa to about275 aa, from about 275 aa to about 300 aa, from about 300 aa to about325 aa, from about 325 aa to about 350 aa, or from about 350 aa to about365 aa. In some cases, an HCV E2 polypeptide suitable for inclusion in avariant E2 polypeptide of an E1/E2 heterodimer of the present disclosureis an HCV E2 ectodomain polypeptide. In some cases, an HCV E2polypeptide suitable for inclusion in a variant E2 polypeptide of anE1/E2 heterodimer of the present disclosure is a full-length HCV E2polypeptide.

In FIG. 1A-AC, the amino acid sequence of E2 is amino acid 384 to aminoacid 746. In FIG. 2A-2B, the amino acid sequence of E2 is amino acid 384to amino acid 751. In FIG. 3A-3C, the amino acid sequence of E2 is aminoacid 385 to amino acid 754. In FIG. 4A-4B, the amino acid sequence of E2is amino acid 384 to amino acid 750. As used herein, an “E2 polypeptide”includes a precursor E2 protein, including the signal sequence; includesa mature E2 polypeptide which lacks this sequence; and includes an E2polypeptide with a heterologous signal sequence. An E2 polypeptide caninclude a C-terminal membrane anchor sequence which occurs atapproximately amino acid positions 715-730 and may extend as far asapproximately amino acid residue 746 (see, Lin et al., J. Virol. (1994)68:5063-5073).

In some cases, a E2 polypeptide suitable for inclusion in a variant E2polypeptide of an E1/E2 heterodimer of the present disclosure lacks aportion of its C-terminal region, e.g., from about amino acid 715 to theC-terminus; from about amino acid 625 to the C-terminus; from aboutamino acid 661 to the C-terminus; from about amino acid 655 to theC-terminus; from about amino acid 500 to the C-terminus, where the aminoacid numbering is with reference to the numbering in FIG. 1A-1C. See,e.g., U.S. Pat. No. 6,521,423.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to an amino acid sequence of an E2polypeptide depicted in FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG.4A-4B. An E2 polypeptide suitable for inclusion in a variant E2polypeptide of an E1/E2 heterodimer of the present disclosure cancomprise an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, or at least about 75%, amino acid sequence identity to anamino acid sequence of an E2 polypeptide depicted in FIG. 1A-1C, FIG.2A-2C, FIG. 3A-3C, or FIG. 4A-4B.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to an amino acid sequence of an E2polypeptide depicted in FIG. 1A-1C. For example, an E2 polypeptide ofgenotype 1 can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 384-746 of an amino acid sequence depicted in FIG. 1A-1C. Forexample, an E2 polypeptide of genotype 1A can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 384-746 of an amino acid sequenceidentified as 1A and depicted in FIG. 1A-1C. For example, an E2polypeptide of genotype 1B can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 384-746 of an amino acid sequence identified as1B and depicted in FIG. 1A-1C. For example, an E2 polypeptide ofgenotype 1C can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 384-746 of an amino acid sequence identified as 1C and depicted inFIG. 1A-1C.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to an amino acid sequence of an E2polypeptide depicted in FIG. 2A-2C. For example, an E2 polypeptide cancomprise an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 384-751of an amino acid sequence depicted in FIG. 2A-2C. For example, an E2polypeptide of genotype 2A can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 384-751 of the “consensus” amino acid sequencedepicted in FIG. 2A-2C. For example, an E2 polypeptide of genotype 2Bcan comprise an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 384-751of the “consensus” amino acid sequence depicted in FIG. 2A-2C.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to an amino acid sequence of an E2polypeptide depicted in FIG. 3A-3C. For example, an E2 polypeptide ofgenotype 3 can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 385-754 of an amino acid sequence depicted in FIG. 3A-3C. Forexample, an E2 polypeptide of genotype 3A can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 385-754 of an amino acid sequenceidentified as 3A and depicted in FIG. 3A-3C. For example, an E2polypeptide of genotype 3B can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 385-754 of the amino acid sequence identified as3B and depicted in FIG. 3A-3C. For example, an E2 polypeptide ofgenotype 3K can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 385-754 of the amino acid sequence identified as 3K and depictedin FIG. 3A-3C.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the amino acid sequence of the E2polypeptide depicted in FIG. 4A-4B. For example, an E2 polypeptide ofgenotype 7A can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 384-750 of the amino acid sequence depicted in FIG. 4A-4B.

Heterologous Polypeptide

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, or more than 10 (e.g., from 10 to 15, from 15 to 20,from 20 to 25, or from 25 to 30, or more than 30), T cell epitopes.T-cell epitopes are epitopes that, when presented with a majorhistocompatibility complex (MHC) (e.g., a human leukocyte antigen (HLA))Class I or MHC Class II molecule, are recognized and bound by a T-cellreceptor (TCR) present on a T cell surface. T-cell epitopes includeepitopes recognized by cytotoxic T cells (e.g., CD8⁺ T cells), andepitopes recognized by helper T cells (e.g., CD4⁺ T cells).

The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) anHCV NS4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NSSApolypeptide; f) an HCV NSSB polypeptide; g) an HCV core polypeptide; orh) an HCV p7 polypeptide. In some cases, the one or more T-cell epitopesare T-cell epitopes present in an HCV NS3 polypeptide. In some cases,the heterologous polypeptide further comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) ameningococcal outer membrane protein. A suitable source of T-cellepitopes non-toxic mutants of toxins, where the mutants are referred toas “cross-reactive material (CRM).” Other examples of strong T helperepitopes are diphtheria toxoid, tetanus toxoid, meningococcal outermembrane protein, or mutant diphtheria protein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127).

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS3 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS3 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS3 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS3 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS3 CD4⁺ T cell epitope and at least one HCV-NS3 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS3 CD4⁺ T-cell epitopes and 2 or more HCV-NS3 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS3 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS3 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS2 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS2 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS2 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS2 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS2 CD4⁺ T cell epitope and at least one HCV-NS2 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS2 CD4⁺ T-cell epitopes and 2 or more HCV-NS2 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS2 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS2 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS4A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS4A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS4A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS4A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS4A CD4⁺ T cell epitope and at least one HCV-NS4A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS4A CD4⁺ T-cell epitopes and 2 or more HCV-NS4A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS4A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS4A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5A CD4⁺ T cell epitope and at least one HCV-NS5A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5A CD4⁺ T-cell epitopes and 2 or more HCV-NS5A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5B T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5B T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5B T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5B T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5B CD4⁺ T cell epitope and at least one HCV-NS5B CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5B CD4⁺ T-cell epitopes and 2 or more HCV-NS5B CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5B CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5B CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-core T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-core T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-core T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-core CD4⁺ T cell epitope and at least one HCV-core CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-core CD4⁺ T-cell epitopes and 2 or more HCV-core CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-core CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-core CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-p7 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-p7 T-cell epitopes.In some cases, the heterologous polypeptide comprises 4 or more HCV-p7T-cell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD4⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core CD4⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises one or more HCV CD8⁺ Tcell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD8⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 CD8⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope. In some cases,the heterologous polypeptide comprises at least one HCV-p7 CD4⁺ T cellepitope and at least one HCV-p7 CD8⁺ T cell epitope. In some cases,heterologous polypeptide comprises 2 or more HCV-p7 CD4⁺ T-cell epitopesand 2 or more HCV-p7 CD8⁺ T-cell epitopes. In some cases, theheterologous polypeptide comprises 2, 3, 4, or 5 HCV-p7 CD4+ T-cellepitopes and 2, 3, 4, or 5 HCV-p7 CD8⁺ T-cell epitopes.

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, or 63, of the T-cell epitopes set out in FIG. 13A-13B. In somecases, the heterologous polypeptide comprises from 1 to 3, from 3 to 5,from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, or from 25 to30 of the T-cell epitopes set out in FIG. 13A-13B. For example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS3-3, NS3-4, and NS3-11 in FIG. 13A-13B and FIG. 15A-15N. Asanother example, in some cases, the heterologous polypeptide comprisesthe T-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 inFIG. 13A-13B and FIG. 15A-15N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-3,NS3-4, NS3-5, and NS3-11 in FIG. 13A-13B and FIG. 15A-15N. As anotherexample, in some cases, the heterologous polypeptide comprises theT-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated Core-1, Core-2, Core-3, Core-4, Core-5, Core-6,Core-7, Core-8, Core-9, Core-10, Core-11, Core-12, Core-13, Core-14,Core-16, Core-17, Core-18, Core-19, Core-20, Core-21, and Core-22 inFIG. 13A-13B and FIG. 15A-15N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-1,NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6, NS2-7, NS2-8,NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1,NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, andNS4b-10 in FIG. 13A-13B and FIG. 15A-15N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8,NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2,NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1,NS5a-2, NS5b-1, and NS5b-2 in FIG. 13A-13B and FIG. 15A-15N. In somecases, the T-cell epitopes are contiguous. In some cases, any two T-cellepitopes are separated by linkers (e.g., a linker having a length offrom 1 amino acid to about 50 amino acids, e.g., from 1 amino acid to 5amino acids (aa), from 5 aa to 10 aa, from 10 aa to 15 aa, from 15 aa to20 aa, from 20 aa to 25 aa, from 25 aa to 30 aa, from 30 aa to 40 aa, orfrom 40 aa to 50 aa).

In some cases, the heterologous polypeptide comprises at least one HCVCD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope, whereepitopes are conserved among HCV genotypes 1 and 2. In some cases, theheterologous polypeptide comprises at least one HCV CD4⁺ T cell epitopeand at least one HCV CD8⁺ T cell epitope, where epitopes are conservedamong HCV genotypes 1 and 3. In some cases, the heterologous polypeptidecomprises at least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺T cell epitope, where epitopes are conserved among HCV genotypes 1, 2,and 3. In some cases, the heterologous polypeptide comprises at leastone HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope,where epitopes are conserved among HCV genotypes 1, 2, 3, and 7. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope, where epitopesare conserved among HCV genotypes 1-7.

The heterologous polypeptide can have a length of from about 10 aminoacids to about 2000 amino acids; e.g., the heterologous polypeptide canhave a length of from 10 amino acids (aa) to 15 aa, from 15 aa to 20 aa,from 20 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to 75 aa, from 75aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aato 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to400 aa, from 450 aa to 500 aa, from 500 aa to 550 aa, from 550 aa to 600aa, from 600 aa to 650 aa, from 650 aa to 700 aa, from 700 aa to 750 aa,or from 750 aa to 800 aa. The heterologous polypeptide can have a lengthof from about 25 amino acids to about 2000 amino acids, e.g., from about25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa,from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aa to 250 aa,from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to 400 aa,from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to 700 aa,from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to 1000 aa,from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aa to 1300aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500 aa to1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from 1800 aato 1900 aa, or from 1900 aa to 2000 aa. The heterologous polypeptide canhave a length of from about 25 amino acids to about 3000 amino acids,e.g., from about 25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aato 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to700 aa, from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to1000 aa, from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aato 1300 aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500aa to 1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from1800 aa to 1900 aa, from 1900 aa to 2000 aa, from 2000 aa to 2250 aa,from 2250 aa to 2500 aa, from 2500 aa to 2750 aa, or from 2750 aa to3000 aa.

The heterologous polypeptide can have a length of from about 25 aminoacids to about 800 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from about 25 aminoacids to about 400 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, or from 350 aa to 400 aa. The heterologouspolypeptide can have a length of 25 amino acids (aa), 26 aa, 27 aa, 28aa, 29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa, 36 aa, 37 aa, 38aa, 39 aa, 40 aa, 41 aa, 42 aa, 43 aa, 44 aa, 45 aa, 46 aa, 47 aa, 48aa, 49 aa, or 50 aa. The heterologous polypeptide can have a length offrom about 100 amino acids (aa) to 800 aa, e.g., from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from 25 aa to 30 aa.The heterologous polypeptide can have a length of from 30 aa to 40 aa.The heterologous polypeptide can have a length of from 40 aa to 50 aa.The heterologous polypeptide can have a length of from 50 aa to 60 aa(e.g., 50 aa, 51 aa, 52, aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa,59 aa, or 60 aa). The heterologous polypeptide can have a length of from60 aa to 70 aa. The heterologous polypeptide can have a length of from65 aa to 75 aa (e.g., 65, 66, 67, 68, 69, 70, 71, 72, 7, 74, or 75 aa).The heterologous polypeptide can have a length of 70 aa. Theheterologous polypeptide can have a length of from 70 aa to 80 aa. Theheterologous polypeptide can have a length of from 80 aa to 90 aa. Theheterologous polypeptide can have a length of from 90 aa to 100 aa. Theheterologous polypeptide can have a length of from 100 aa to 105 aa(e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 aa). Theheterologous polypeptide can have a length of 100 aa. The heterologouspolypeptide can have a length of from 10 amino acids (aa) to 50 aa;e.g., from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to 25 aa,from 25 aa to 30 aa, from 30 aa to 35 aa, from 35 aa to 40 aa, from 40aa to 45 aa, or from 45 aa to 50 aa. The heterologous polypeptide canhave a length of from 10 amino acids (aa) to 20 aa, e.g., 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 aa.

HCV NS3 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS3 polypeptide. Examplesof T-cell epitopes present in NS3 polypeptides are depicted in FIG.15A-15N, FIG. 13B, and FIG. 14A-14B.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1). In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1); and has a length of from 25aa to 35 aa (e.g., 25 aa, 26 aa, 27 aa, 28 aa, 29 aa, 30 aa, 31 aa, 32aa, 33 aa, 34 aa, or 35 aa). In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1); and has alength of 29 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-3, NS3-4, and NS3-11 in FIG. 13B and FIG.15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2); andhas a length of from 45 amino acids to 60 amino acids (e.g., 45 aa, 46aa, 47 aa, 48 aa, 49 aa, 50 aa, 51 aa, 52 aa, 53 aa, 54 aa, 55 aa, 56aa, 57 aa, 58 aa, 59 aa, or 60 aa). In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2); andhas a length of 52 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-3, NS3-4, NS3-5, and NS3-11 in FIG. 13B and FIG.15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:3); and has a length of from 65 amino acids to 80 amino acids(e.g., 65 aa, 66 aa, 67 aa, 68 aa, 69 aa, 70 aa, 71 aa, 72 aa, 73 aa, 74aa, 75 aa, 76 aa, 77 aa, 78 aa, 79 aa, or 80 aa). In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:3); and has a length of 70 amino acids. Such a polytope caninclude NS3 T-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6,NS3-7, NS3-11, NS3-12, and NS3-13 in FIG. 13B and FIG. 15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4); and has a lengthof from 95 amino acids (aa) to 105 aa (e.g., 95 aa, 96 aa, 97 aa, 98 aa,99 aa, 100 aa, 101 aa, 102 aa, 103 aa, 104 aa, or 105 aa). In somecases, the heterologous polypeptide comprises an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4); and has a lengthof 100 amino acids. Such a polytope can include NS3 T-cell epitopesdesignated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 13B and FIG. 15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:9); and has a length of from 190 aminoacids (aa) to 200 aa (e.g., 190 aa, 191 aa, 192 aa, 193 aa, 194 aa, 195aa, 196 aa, 197 aa, 198 aa, 199 aa, or 200 aa. In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:9); and has a length of 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:10); and hasa length of from 215 amino acids (aa) to 235 aa (e.g., 215 aa, 216 aa,217 11, 218 aa, 219 aa, 220 aa, 221 aa, 222 aa, 223 aa, 224 aa, 225 aa,226 aa, 227 aa, 228 aa, 229 aa, 230 aa, 231 aa, 232 aa, 233 aa, 234 aa,or 235 aa). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:10); and hasa length of 228 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 13B and FIG. 15A-15N.

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1265-1279 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1309-1323 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1401-1415 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1402-1412 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1429-1439 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1464 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1453-1467 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1577-1591 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1306-1314 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1387-1394 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 1amino acids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12aa, 13 aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1405-1413 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1458 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1457-1465 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1610-1618 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

HCV NS2 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS2 polypeptide. Examplesof T-cell epitopes present in NS2 polypeptides are depicted in FIG.15A-15N, and FIG. 13A.

For example, the heterologous polypeptide can comprise an NS2 T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids955-974 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 975-994 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 985-1004 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1015-1034 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1035-1054 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 924-933 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 961-970 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 989-997 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 50 aa (e.g., from 10 aa to 25 aa, or from 25 aa to 50 aa) ofamino acids 955-1004 of the amino acid sequence designated “Consensus”in FIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa, orfrom 25 aa to 50 aa. In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 955-1004of the amino acid sequence designated “Consensus” in FIG. 16A-16L, or acorresponding HCV NS2 amino acid sequence of any HCV genotype; and has alength of about 50 amino acids.

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 553 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from 300 aato 400 aa, from 400 aa to 500 aa, or from 500 aa to 553 aa) of aminoacids 917-1469 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 and NS3 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa,from 25 aa to 50 aa, from 50 aa to 100 aa, from 100 aa to 200 aa, from200 aa to 300 aa, from 300 aa to 400 aa, from 400 aa to 500 aa, or from500 aa to 553 aa. In some cases, the heterologous polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 917-1469 of the aminoacid sequence designated “Consensus” in FIG. 16A-16L, or a correspondingHCV NS2 and NS3 amino acid sequence of any HCV genotype; and has alength of about 553 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 0%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11); andhas a length of from 50 amino acids to 60 amino acids (e.g., 50 aa, 51aa, 52 aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60 aa).In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11); andhas a length of 50 amino acids. Such a polytope can include NS2 T-cellepitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 in FIG. 13Aand FIG. 15A-15N.

HCV NS4A T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS4A polypeptide. Examplesof T-cell epitopes present in NS4A polypeptides are depicted in FIG.15A-15N and FIG. 13B.

The heterologous polypeptide can comprise an NS4A T cell epitopecomprising an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids1683-1692 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS4A amino acid sequence of any HCVgenotype; and the NS4A T-cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

HCV NS4B T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS4B polypeptide. Examplesof T-cell epitopes present in NS4B polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NS4B T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids1790-1801 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 12 aminoacids (aa) to 20 amino acids (e.g., 12 aa, 13 aa, 14 aa, 15 aa, 16 aa,17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1792-1802 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 11 aminoacids (aa) to 20 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15 aa,16 aa, 17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1898-1905 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 8 aminoacids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1921-1935 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1922-1941 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1928-1947 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1868-1876 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1927-1942 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 16 aminoacids (aa) to 20 amino acids (e.g., 16 aa, 17 aa, 18 aa, 19 aa, or 20aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1932-1940 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1948-1962 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

HCV NSSA T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NSSA polypeptide. Examplesof T-cell epitopes present in NSSA polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NSSA T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2218-2232 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS5A amino acid sequence of any HCVgenotype; and the NS5A T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NS5A Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2309-2317 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS5A amino acid sequence of any HCVgenotype; and the NS5A T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV NS5B T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS5B polypeptide. Examplesof T-cell epitopes present in NS5B polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NSSB T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2847-2851 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NSSB amino acid sequence of any HCVgenotype; and the NSSB T-cell epitope can have a length of from 5 aminoacids (aa) to 10 amino acids (e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10aa).

As another example, the heterologous polypeptide can comprise an NSSB Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2602-2610 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NSSB amino acid sequence of any HCVgenotype; and the NSSB T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV Core T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV core polypeptide. Examplesof T-cell epitopes present in HCV Core polypeptides are depicted in FIG.15A-15N and FIG. 13A.

As one example, the heterologous polypeptide can comprise an HCV core Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1-20 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 11-30 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 21-40 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 39-63 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 23amino acids (aa) to 28 amino acids (e.g., 23 aa, 24 aa, 25 aa, 26 aa, 27aa, or 28 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 47-70 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 24amino acids (aa) to 29 amino acids (e.g., 24 aa, 25 aa, 26 aa, 27 aa, 28aa, or 29 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 61-80 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 71-90 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 81-100 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 91-110 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 101-115 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 111-130 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 125-139 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-150 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 151-170 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 161-180 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 35-44 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 43-51 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 51-59 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 129-137 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-140 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 150-158 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 154-162 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 168-176 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 177-187 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 178-187 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 191 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, or from 150 aato 191 aa) of amino acids 1-191 of the amino acid sequence designated“Consensus” in FIG. 16A-16L, or a corresponding HCV core amino acidsequence of any HCV genotype; and has a length of from 10 amino acids(aa) to 25 aa, from 25 aa to 50 aa, from 50 aa to 100 aa, or from 100 aato 150 aa, or from 150 aa to 191 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to amino acids1-191 of the amino acid sequence designated “Consensus” in FIG. 16A-16L,or a corresponding HCV core amino acid sequence of any HCV genotype; andhas a length of about 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLE DGVNYATGNLPG (SEQID NO:63); and has a length of from 171 amino acids (aa) to 180 aa(e.g., 171 aa, 172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa,179 aa, or 180 aa. In some cases, the heterologous polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLE DGVNYATGNLPG (SEQID NO:63); and has a length of 171 amino acids. Such a polytope caninclude core T-cell epitopes designated Core-1, Core-2, Core-3, Core-4,Core-5, Core-6, Core-7, Core-8, Core-9, Core-10, Core-11, Core-12,Core-13, Core-14, Core-16, Core-17, Core-18, Core-19, Core-20, Core-21,Core-22 in FIG. 13A and FIG. 15A-15N.

HCV p7 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV p7 polypeptide. Examplesof T-cell epitopes present in HCV p7 polypeptides are depicted in FIG.15A-15N or FIG. 13A.

As another example, the heterologous polypeptide can comprise an HCV p7T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 803-811 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV p7 amino acid sequence of any HCVgenotype; and the HCV p7 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

Polytopes Including HCV T-Cell Epitopes from More than One HCVPolypeptide Other than E1 and E2

As noted above, a heterologous polypeptide can include T-cell epitopesfrom more than one HCV polypeptide other than E1 and E2.

As one example, a heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTG DFDSVIDCN (SEQ IDNO:12); and has a length of from 550 amino acids (aa) to 560 aa (e.g.,550 aa, 551 aa, 552 aa, 553 aa, 554 aa, 555 aa, 556 aa, 557 aa, 558 aa,559 aa, or 560 aa). In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTG DFDSVIDCN (SEQ IDNO:12); and has a length of 553 amino acids. Such a polytope can includeT-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6,NS2-7, NS2-8, NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9,NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N.This polytope is also referred to as “TP553” (FIG. 14A-14D). In order toprevent self cleavage of the TP553 polytope (amino acids 917-1469) (FIG.15E-G) at the NS2-NS3 junction that is mediated by the catalytic domainof the NS2 protease (amino acids 917-1040), the histidine at position966 (H966), a critical residue for NS2 protease activity, is mutated toalanine (H966A) (FIG. 15E). See, e.g., Grakoui, A. et al. A secondhepatitis C virus-encoded proteinase. Proc. Natl Acad. Sci. USA 90,10583-10587 (1993); Hijikata, M. et al. Two distinct proteinaseactivities required for the processing of a putative nonstructuralprecursor protein of hepatitis C virus. J. Virol. 67, 4665-4675 (1993);and Lorenz. IC. Structure of the catalytic domain of the hepatitis Cvirus NS2-3 protease. Nature. August 17; 442(7104):831-5 (2006).

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aato 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa) the followingamino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to a contiguous stretch of from 25amino acids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa,from 200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa,from 350 aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa)of the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64); and has a length of from 25 amino acids (aa) to 50 aa,from 50 aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from300 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600aa to 700 aa, or from 700 aa to 778 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64); and has a length of 778 amino acids. Such a polytope caninclude T-cell epitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5,NS3-6, NS3-7, NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS2-14,NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8,NS4b-9, and NS4b-10 in FIG. 13B and FIG. 15A-15N.

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 1985 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 500 aa, from 500 aa to 750 aa, from 750aa to 1000 aa, from 1000 aa to 1500 aa, or from 1500 aa to 1985 aa) ofthe following amino acid sequence:

(SEQ ID NO: 13) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTV YHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVT RHADVIPVRRRGDSRGSLLSPRPISYLKGS A GGPLLCPAGHAVGIFRAAVCTRGV AKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAA YAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYG KFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPP GSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLV ALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQT VDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSV LCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHID AHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPT PLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCL STGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQ KALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPG NPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGA AIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGAL VVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARV TAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKL MPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTC RNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGM TTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQL PCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCT ANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVP AEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPP RKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAE SYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAE EQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEV KAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVW KDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYD VVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTE SDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVL TTSCGNTLTCYIKARAACRAAGLQDCTMLVCG NN LVVICESAGVQEDAASLRA FTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLA RAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEI YGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHR ARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR. 

In some cases, the heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGMTTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPPRKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGNNLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR (SEQ ID NO:13); and has a length of1985 amino acids. Such a polytope can include T-cell epitopes designatedNS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8, NS3-9, NS3-10,NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4,NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1, NS5a-2, NS5b-1,NS5b-2 in FIG. 13A-13B and FIG. 15A-15N.

Additional T-Cell Epitopes

As discussed above, an E1/E2 a heterodimeric polypeptide of the presentdisclosure includes: a) an HCV E1 polypeptide; and b) a variant HCV E2polypeptide comprising: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide). The one or more T-cell epitopescan include one or more T-cell epitopes present in: a) an HCV NS3polypeptide; b) an HCV NS2 polypeptide; c) an HCV NS4A polypeptide; d)an HCV NS4B polypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5Bpolypeptide; g) an HCV core polypeptide; or h) an HCV p7 polypeptide. Insome cases, the one or more T-cell epitopes are T-cell epitopes presentin an HCV NS3 polypeptide. In some cases, the heterologous polypeptidefurther comprises one or more T cell epitopes present in: a) choleratoxin or toxoid; and/or b) tetanus toxin or toxoid; and/or c) diphtheriatoxin or toxoid; and/or d) a meningococcal outer membrane protein.

Thus, in some cases, a variant HCV E2 polypeptide of an E1/E2heterodimer of the present disclosure includes: a) an HCV E2polypeptide; and b) a heterologous polypeptide that comprises one ormore T-cell epitopes, where the one or more T-cell epitopes are T-cellepitopes present in: i) one or more of an HCV NS3 polypeptide, an HCVNS2 polypeptide, an HCV NS4A polypeptide, an HCV NS4B polypeptide, anHCV NS5A polypeptide, an HCV NS5B polypeptide, an HCV core polypeptide,and an HCV p7 polypeptide; and ii) one or more of cholera toxin ortoxoid, tetanus toxin or toxoid, diphtheria toxin or toxoid, and ameningococcal outer membrane protein.

A T helper tetanus toxin epitope or other bacterial T-cell epitope couldbe fused (e.g., by recombinant expression) or chemically conjugated tothe HCV polytope/E2 fusion protein and/or to the HCV polytope E1 fusionprotein of an E/E2 heterodimer of the present disclosure to furtherenhance both T and B cell responses to both the HCV polytope and E1/E2moieties. Alternatively, the whole or part of the detoxified toxin(“toxoid”) could be fused (e.g., by recombinant expression) orchemically conjugated to the HCV polytope/E1E2 protein, wherein specificamino acids of the toxins are mutated to render the toxins inactive,thereby generating toxoids. Methods of generating toxoids are well knownin the art. Examples of bacterial epitopes include the use of diphtheriatoxoid, meningococcal outer membrane protein, or mutant diphtheriaprotein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127)

In some cases, a suitable tetanus toxoid polypeptide comprises the aminoacid sequence QYIKANSKFIGIFE (SEQ ID NO:14). In some cases, a suitabletetanus toxoid polypeptide comprises the amino acid sequenceQYIKANSKFIGITE (SEQ ID NO:65).

In some cases, a heterologous polypeptide can comprise cholera toxin (ortoxoid) epitope. In some cases, a suitable heterologous polypeptidecomprising a cholera toxoid epitope comprises a fragment of choleratoxin-B subunit (CT-B), e.g., a fragment of from 5 amino acids to 25amino acids, or from 25 amino acids to 50 amino acids, of the followingamino acid sequence: MIKLKFGVFF TVLLSSAYAH GTPQNITDLC AEYHNTQIHTLNDKIFSYTE SLAGKREMAI ITFKNGATFQ VEVPGSQHID SQKKAIERMK DTLRIAYLTEAKVEKLCVWN NKTPHAIAAI SMAN (SEQ ID NO:15).

In some cases, a heterologous polypeptide can comprise a tetanus toxin(or toxoid) T-cell epitope. In some cases, a suitable heterologouspolypeptide comprising a tetanus toxin T-cell epitope comprises theamino acid sequence: ILMQYIKANSKFIGI (SEQ ID NO:16); and has a length offrom 15 amino acids to 20 amino acids. In some cases, a suitableheterologous polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: VNNESSE (SEQ ID NO:17). In somecases, a suitable heterologous polypeptide comprising a tetanus toxinT-cell epitope comprises the amino acid sequence: PGINGKAIHLVNNESSE (SEQID NO:18). In some cases, a suitable heterologous polypeptide comprisinga tetanus toxin T-cell epitope comprises the amino acid sequence: PNRDIL(SEQ ID NO:19). In some cases, a suitable heterologous polypeptidecomprising a tetanus toxin T-cell epitope comprises the amino acidsequence: FIGITEL (SEQ ID NO:20). In some cases, a suitable tetanustoxin T-cell epitope comprises the amino acid sequence: SYFPSV (SEQ IDNO:21). In some cases, a suitable heterologous polypeptide comprising atetanus toxin T-cell epitope comprises the amino acid sequence:NSVDDALINSTKIYSYFPSV (SEQ ID NO:22). In some cases, a suitableheterologous polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: IDKISDVSTIVPYIGPALNI (SEQ ID NO:23).

In some cases, a heterologous polypeptide can comprise a diphtheriatoxin T-cell epitope In some cases, a suitable heterologous polypeptidecomprising a diphtheria toxin T-cell epitope comprises the amino acidsequence: QSIALSSLMVAQAIP (SEQ ID NO:24); and has a length of from 15amino acids to 20 amino acids. In some cases, a suitable heterologouspolypeptide comprising a diphtheria toxin T-cell epitope comprises theamino acid sequence: PVFAGANYAAWAVNVAQVI (SEQ ID NO:25). In some cases,a suitable heterologous polypeptide comprising a diphtheria toxin T-cellepitope comprises the amino acid sequence: VHHNTEEIVAQSIALSSLMV (SEQ IDNO:26). In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QSIALSSLMVAQAIPLVGEL (SEQ ID NO:66). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: VDIGFAAYNFVESIINLFQV (SEQ ID NO:67).In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QGESGHDIKITAENTPLPIA (SEQ ID NO:68). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: GVLLPTIPGKLDVNKSKTHI (SEQ ID NO:69).In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence ofCRM197 (see, e.g., Giannini et al. (1984) Nucl. Acids. Res. 12:4063).

The amino acid sequence of CRM197 is as follows:

1addvvdssksfvmenfssyhgtkpgyvdsigkgigkpksgtqgnydddwkefystdnkydaagysvdnenplsgkaggvvkvtypgltkvlalkvdnaetikkelglslteplmeqvgteefikrfgdgasrvvlslpfaegsssveyinnwegakalsveleinfetrgkrgqdamyeymaqacagnrvrrsvgsslscinldwdvirdktktkieslkehgpiknkmsespnktvseekakqyleefhqtalehpelselktvtgtnpvfaganyaawavnvaqvidsetadnlekttaalsilpgigsvmgiadgavhhnteeivaqsialsslmvaqaiplvgelvdigfaaynfvesiinlfqvvhnsynrpayspghktqpflhdgyavswntvedsiirtgfqgesghdikitaentplpiagvllptipgkldvnkskthisvngrkirmrcraidgdvtfcrpkspvyvgngvhanlhvafhrsssekihsneissdsigvlgyqktvdhtkvnsklslffeiks (SEQID NO:27).

In some cases, a heterologous polypeptide can comprise a tetanus toxinT-cell epitope and a diphtheria toxin T-cell epitope. In some of thesecases, the heterologous polypeptide can comprise the amino acidsequence: IMQYIKANSKFIGIQSIALSSLMVAQ (SEQ ID NO:28); and can have alength of from 26 amino acids to 30 amino acids.

E1

An HCV E1 polypeptide suitable for inclusion in an E1/E2 heterodimer ofthe present disclosure can have a length of from about 150 amino acids(aa) to about 175 aa, from about 175 aa to about 195 aa, from about 131aa to about 175 aa, or from about 175 aa to about 193 aa. In some cases,an HCV E1 polypeptide suitable for inclusion in an E1/E2 heterodimer ofthe present disclosure is an HCV E1 ectodomain polypeptide. In somecases, an HCV E1 polypeptide suitable for inclusion in an E1/E2heterodimer of the present disclosure is a full-length HCV E1polypeptide.

In FIG. 1A-1C, the amino acid sequence of E1 is amino acid 192 to aminoacid 383. In FIG. 2A-2C, the amino acid sequence of E1 is amino acid 192to amino acid 383. In FIG. 3A-3C, the amino acid sequence of E1 is aminoacid 192 to amino acid 384. In FIG. 4A-4B, the amino acid sequence of E1is amino acid 192 to amino acid 383. Amino acids at around 170 throughapproximately 191 serve as a signal sequence for E1. As used herein, “E1polypeptide” includes a precursor E1 protein, including the signalsequence; includes a mature E1 polypeptide which lacks this sequence;and includes an E1 polypeptide with a heterologous signal sequence. AnE1 polypeptide can include a C-terminal membrane anchor sequence whichoccurs at approximately amino acid positions 360-383 (see, e.g., WO96/04301). In some cases, a suitable E1 polypeptide lacks a C-terminalportion that includes a transmembrane region. For example, in somecases, a suitable E1 polypeptide lacks the C-terminal portion from aminoacid 330 to amino acid 384, or from amino acid 360 to amino acid 384. E1polypeptides can be an E1 polypeptide of any genotype, subtype orisolate of HCV. E1 polypeptides of genotype 1 and E1 polypeptides ofgenotype 3 are included in an E1/E2 heterodimer of the presentdisclosure.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 1A-1C, FIG.2A-2C, FIG. 3A-3C, or FIG. 4A-4B.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 1A-1C. Forexample, an E1 polypeptide of genotype 1A can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 192-383 of an amino acid sequenceidentified as 1A and depicted in FIG. 1A-1C. For example, an E1polypeptide of genotype 1B can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 192-383 of an amino acid sequence identified as1B and depicted in FIG. 1A-1C. For example, an E1 polypeptide ofgenotype 1C can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 192-383 of an amino acid sequence identified as 1C and depicted inFIG. 1A-1C.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 2A-2C. Forexample, an E1 polypeptide of genotype 2A can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 192-383 of an amino acid sequenceidentified as 2A and depicted in FIG. 2A-2C. For example, an E1polypeptide of genotype 2B can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 192-383 of an amino acid sequence identified as2B and depicted in FIG. 2A-2C.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity tothe consensus E1 polypeptide amino acid sequence depicted in FIG. 3A-3C.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 4A-4B. Forexample, an E1 polypeptide of genotype 7A can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 192-383 of the amino acid sequencedepicted in FIG. 4A-4B.

Additional Polypeptides

In any of the above-described embodiments, one or both of thepolypeptide chains of the E1/E2 heterodimer can include one or moreadditional polypeptides. For example, in some cases, the E1 polypeptideor the variant E2 polypeptide can include an Ig Fc polypeptide at theC-terminus of the E1 polypeptide or the variant E2 polypeptide. Asanother example, in some cases, the E1 polypeptide or the variant E2polypeptide can include an Ig Fc polypeptide at the N-terminus of the E1polypeptide or the variant E2 polypeptide. Ig Fc polypeptides are knownin the art, and are described elsewhere herein.

IB. E1E2 Heterodimers Comprising a Variant HCV E1 and HCV E2

The present disclosure provides an E1/E2 heterodimer, where the E1/E2heterodimer comprises: a) a variant HCV E1 polypeptide, where thevariant HCV E1 polypeptide comprises: i) an HCV E1 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T-cell epitopes not present in an HCV E1 or an HCV E2polypeptide; e.g., one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 or an HCV E2 polypeptide); and b) anHCV E2 polypeptide. Thus, in some cases, a heterodimeric polypeptide ofthe present disclosure includes: a) an HCV E2 polypeptide; and b) avariant HCV E1 polypeptide comprising: i) an HCV E1 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). The one or moreT-cell epitopes can include one or more T-cell epitopes present in: a)an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) an HCV NS4Apolypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5A polypeptide; f)an HCV NS5B polypeptide; g) an HCV core polypeptide; or h) an HCV p7polypeptide. In some cases, the one or more T-cell epitopes are T-cellepitopes present in an HCV NS3 polypeptide. In some cases, theheterologous polypeptide further comprises one or more T cell epitopespresent in: a) cholera toxin or toxoid; and/or b) tetanus toxin ortoxoid; and/or c) diphtheria toxin or toxoid; and/or d) a meningococcalouter membrane protein. The heterologous polypeptide is also referred toas a “polytope.”

An E1/E2 heterodimer of the present disclosure (e.g., an E1/E2heterodimer comprising: a) an HCV E2 polypeptide; and b) a variant HCVE2 polypeptide comprising: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes), whenadministered to an individual in need thereof, induces an immuneresponse in the individual to one or more HCV genotypes. An E1/E2heterodimer of the present disclosure, when administered to anindividual in need thereof, induces an immune response in the individualto one or more HCV genotypes, where the immune response is greater thanthe immune response induced by administration of an HCV E1/E2heterodimer comprising a wild-type E2 and a wild-type E1 polypeptide oran E1 polypeptide lacking the polytope.

For example, in some cases, an E1/E2 heterodimer of the presentdisclosure (e.g., an E1/E2 heterodimer comprising: a) an HCV E2polypeptide; and b) a variant HCV E1 polypeptide comprising: i) an HCVE1 polypeptide; and ii) a heterologous polypeptide comprising one ormore T-cell epitopes), when administered to an individual in needthereof, induces CTLs specific for HCV, where the number of HCV-specificCTLs induced is at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 40%, at least 50%, at least 75%, at least 100%(or 2-fold), at least 2.5-fold, at least 5-fold, at least 7.5-fold, atleast 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold,or more than 100-fold, higher than the number of HCV-specific CTLsinduced by administration of an HCV E1/E2 heterodimer comprising awild-type E2 and a wild-type E1 polypeptide or an E1 polypeptide lackingthe polytope.

In some cases, an E1/E2 heterodimer of the present disclosure (e.g., anE1/E2 heterodimer comprising: a) an HCV E2 polypeptide; and b) a variantHCV E1 polypeptide comprising: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces production of HCV-specific CD4⁺ T cells and CD8⁺ T cells in theindividual, where the number of HCV-specific CD4⁺ T cells and CD8⁺ Tcells is increased, such that the percent of the total peripheral bloodT cells (i.e., the total number of CD4⁺ T cells+CD8⁺ T cells in theperipheral blood) that are HCV-specific CD4⁺ T cells and CD8⁺ T cells isfrom 0.5% to 10% (e.g., from 0.5% to 1%, from 1% to 2%, from 2% to 5%,or from 5% to 10%). The number of HCV-specific CD4⁺ T cells and CD8⁺ Tcells in a control individual (e.g., an individual not infected withHCV) not treated with the E1/E2 heterodimer would be undetectable.

For example, in some cases, an E1/E2 heterodimer of the presentdisclosure (e.g., an E1/E2 heterodimer comprising: a) an HCV E2polypeptide; and b) a variant HCV E1 polypeptide comprising: i) an HCVE1 polypeptide; and ii) a heterologous polypeptide comprising one ormore HCV NS3 T-cell epitopes), when administered to an individual inneed thereof, induces production of HCV NS3-specific CD4⁺ T cells andCD8⁺ T cells in the individual, where the number of HCV NS3-specificCD4⁺ T cells and CD8⁺ T cells is increased, such that the percent of thetotal peripheral blood T cells (i.e., the total number of CD4⁺ Tcells+CD8⁺ T cells in the peripheral blood) that are HCV NS3-specificCD4⁺ T cells and CD8⁺ T cells is from 0.1% to 10% (e.g., from 0.1% to0.5%, from 0.5% to 1%, from 1% to 2%, from 2% to 5%, or from 5% to 10%).The number of HCV NS3-specific CD4⁺ T cells and CD8⁺ T cells in acontrol individual (e.g., an individual not infected with HCV) nottreated with the E1/E2 heterodimer would be undetectable.

In some cases, an E1/E2 heterodimer of the present disclosure (e.g., anE1/E2 heterodimer comprising: a) an HCV E2 polypeptide; and b) a variantHCV E1 polypeptide comprising: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,increases the number of HCV E1/E2-specific CD4⁺ T cells and CD8⁺ T cellsin the individual by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 40%, at least 50%, at least 75%, at least100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 7.5-fold,at least 10-fold, at least 20-fold, at least 50-fold, or at least100-fold, or more than 100-fold, compared to the number of HCVE1/E2-specific CD4⁺ T cells and CD8⁺ T cells in the individual inducedby administration of an E1/E2 heterodimer comprising a wild-type E2polypeptide and a wild-type E1 polypeptide, or an E1 polypeptide lackingthe polytope, or compared to the number of HCV E1/E2-specific CD4⁺ Tcells and CD8⁺ T cells in the individual before administration of theE1/E2 heterodimer of the present disclosure.

As another example, in some cases, an E1/E2 heterodimer of the presentdisclosure (e.g., an E1/E2 heterodimer comprising: a) an HCV E2polypeptide; and b) a variant HCV E1 polypeptide comprising: i) an HCVE1 polypeptide; and ii) a heterologous polypeptide comprising one ormore T-cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide), when administered to anindividual in need thereof, induces helper T lymphocytes (e.g., CD4⁺ Tcells) specific for HCV, where the number of HCV-specific helper Tlymphocytes induced is at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 75%, atleast 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least7.5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or atleast 100-fold, or more than 100-fold, higher than the number ofHCV-specific helper T cells induced by administration of an E1/E2heterodimer comprising a wild-type E2 polypeptide and a wild-type E1polypeptide, or an E1 polypeptide lacking the polytope, or compared tothe number of HCV-specific helper T cells in the individual beforeadministration of the E1/E2 heterodimer of the present disclosure.

In some cases, an E1/E2 heterodimer of the present disclosure (e.g., anE1/E2 heterodimer comprising: a) an HCV E2 polypeptide; and b) a variantHCV E1 polypeptide comprising: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces antibody specific for HCV, where the level of HCV-specificantibody induced is at least at high as the level of HCV-specificantibody induced by administration of an E1/E2 heterodimer comprising awild-type E2 polypeptide and a wild-type E1 polypeptide, or an E1polypeptide lacking the polytope.

In some cases, an E1/E2 heterodimer of the present disclosure (e.g., anE1/E2 heterodimer comprising: a) an HCV E2 polypeptide; and b) a variantHCV E1 polypeptide comprising: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces antibody specific for HCV, where the level of HCV-specificantibody induced is at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 40%, at least 50%, at least 75%, at least100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 7.5-fold,at least 10-fold, at least 20-fold, at least 50-fold, or at least100-fold, or more than 100-fold, higher than the level of HCV-specificantibody induced by administration of an E1/E2 heterodimer comprising awild-type E2 polypeptide and a wild-type E1 polypeptide, or an E1polypeptide lacking the polytope.

An E1/E2 heterodimer of the present disclosure (e.g., an E1/E2heterodimer comprising: a) an HCV E2 polypeptide; and b) a variant HCVE1 polypeptide comprising: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces an immune response (e.g., a cellular immune response) in theindividual to one or more HCV genotypes. In some cases, an E1/E2heterodimer of the present disclosure (e.g., an E1/E2 heterodimercomprising: a) an HCV E2 polypeptide; and b) a variant HCV E1polypeptide comprising: i) an HCV E1 polypeptide; and ii) a heterologouspolypeptide comprising one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide),when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 1. In some cases, an E1/E2heterodimer of the present disclosure (e.g., an E1/E2 heterodimercomprising: a) an HCV E2 polypeptide; and b) a variant HCV E1polypeptide comprising: i) an HCV E1 polypeptide; and ii) a heterologouspolypeptide comprising one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide),when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 2. In some cases, an E1/E2heterodimer of the present disclosure (e.g., an E1/E2 heterodimercomprising: a) an HCV E2 polypeptide; and b) a variant HCV E1polypeptide comprising: i) an HCV E1 polypeptide; and ii) a heterologouspolypeptide comprising one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide),when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 3. In some cases, an E1/E2heterodimer of the present disclosure (e.g., an E1/E2 heterodimercomprising: a) an HCV E2 polypeptide; and b) a variant HCV E1polypeptide comprising: i) an HCV E1 polypeptide; and ii) a heterologouspolypeptide comprising one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide),when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 1 and HCV genotype 3. In somecases, an E1/E2 heterodimer of the present disclosure (e.g., an E1/E2heterodimer comprising: a) an HCV E2 polypeptide; and b) a variant HCVE1 polypeptide comprising: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces an immune response in the individual to HCV genotype 1, HCVgenotype 2, and HCV genotype 3. In some cases, an E1/E2 heterodimer ofthe present disclosure (e.g., an E1/E2 heterodimer comprising: a) an HCVE2 polypeptide; and b) a variant HCV E1 polypeptide comprising: i) anHCV E1 polypeptide; and ii) a heterologous polypeptide comprising one ormore T-cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide), when administered to anindividual in need thereof, induces an immune response in the individualto HCV genotype 1, HCV genotype 2, HCV genotype 3, and HCV genotype 7.In some cases, an E1/E2 heterodimer of the present disclosure (e.g., anE1/E2 heterodimer comprising: a) an HCV E2 polypeptide; and b) a variantHCV E1 polypeptide comprising: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide comprising one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide), when administered to an individual in need thereof,induces an immune response in the individual to HCV genotype 1, HCVgenotype 2, HCV genotype 3, HCB genotype 4, HCV genotype 5, HCV genotype6, and HCV genotype 7.

Variant E1

As noted above, a variant E1 polypeptide of an HCV E1/E2 heterodimer ofthe present disclosure comprises: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide comprising one or more T cell epitopes (e.g.,one or more T-cell epitopes not present in an HCV E1 or an HCV E2polypeptide; e.g., one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 or an HCV E2 polypeptide). In somecases, the variant HCV E1 polypeptide comprises, in order from aminoterminus (N-terminus) to carboxyl terminus (C-terminus): i) an HCV E1polypeptide; and ii) a heterologous polypeptide comprising one or more Tcell epitopes. In some cases, the variant HCV E1 polypeptide comprises,in order from N-terminus to C-terminus: i) a heterologous polypeptidecomprising one or more T cell epitopes; and ii) an HCV E1 polypeptide.

E1

An HCV E1 polypeptide suitable for inclusion in a variant E1 polypeptideof an E1/E2 heterodimer of the present disclosure can have a length offrom about 150 amino acids (aa) to about 175 aa, from about 175 aa toabout 195 aa, from about 131 aa to about 175 aa, or from about 175 aa toabout 193 aa. In some cases, an HCV E1 polypeptide suitable forinclusion in a variant E1 polypeptide of an E1/E2 heterodimer of thepresent disclosure is an HCV E1 ectodomain polypeptide. In some cases,an HCV E1 polypeptide suitable for inclusion in a variant E1 polypeptideof an E1/E2 heterodimer of the present disclosure is a full-length HCVE1 polypeptide.

In FIG. 1A-1C, the amino acid sequence of E1 is amino acid 192 to aminoacid 383. In FIG. 2A-2C, the amino acid sequence of E1 is amino acid 192to amino acid 383. In FIG. 3A-3C, the amino acid sequence of E1 is aminoacid 192 to amino acid 384. In FIG. 4A-4B, the amino acid sequence of E1is amino acid 192 to amino acid 383. Amino acids at around 170 throughapproximately 191 serve as a signal sequence for E1. As used herein, “E1polypeptide” includes a precursor E1 protein, including the signalsequence; includes a mature E1 polypeptide which lacks this sequence;and includes an E1 polypeptide with a heterologous signal sequence. AnE1 polypeptide can include a C-terminal membrane anchor sequence whichoccurs at approximately amino acid positions 360-383 (see, e.g., WO96/04301). In some cases, a suitable E1 polypeptide lacks a C-terminalportion that includes a transmembrane region. For example, in somecases, a suitable E1 polypeptide lacks the C-terminal portion from aminoacid 330 to amino acid 384, or from amino acid 360 to amino acid 384. E1polypeptides can be an E1 polypeptide of any genotype, subtype orisolate of HCV. E1 polypeptides of genotype 1 and E1 polypeptides ofgenotype 3 are included in an E1/E2 heterodimer of the presentdisclosure.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to an amino acid sequence of an E1polypeptide depicted in FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG.4A-4B.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to an amino acid sequence of an E1polypeptide depicted in FIG. 1A-1C. For example, an E1 polypeptide ofgenotype 1A can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 192-383 of an amino acid sequence identified as 1A and depicted inFIG. 1A-1C. For example, an E1 polypeptide of genotype 1B can comprisean amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 192-383 of an aminoacid sequence identified as 1B and depicted in FIG. 1A-1C. For example,an E1 polypeptide of genotype 1C can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 192-383 of an amino acid sequenceidentified as 1C and depicted in FIG. 1A-1C.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to an amino acid sequence of an E1polypeptide depicted in FIG. 2A-2C. For example, an E1 polypeptide ofgenotype 2A can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 192-383 of an amino acid sequence identified as 2A and depicted inFIG. 2A-2C. For example, an E1 polypeptide of genotype 2B can comprisean amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 192-383 of an aminoacid sequence identified as 2B and depicted in FIG. 2A-2C.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the consensus E1 polypeptide amino acidsequence depicted in FIG. 3A-3C.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to an amino acid sequence of an E1polypeptide depicted in FIG. 4A-4B. For example, an E1 polypeptide ofgenotype 7A can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 192-383 of the amino acid sequence depicted in FIG. 4A-4B.

Heterologous Polypeptide

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, or more than 10 (e.g., from 10 to 15, from 15 to 20,from 20 to 25, or from 25 to 30, or more than 30), T cell epitopes.T-cell epitopes are epitopes that, when presented with a majorhistocompatibility complex (MHC) (e.g., a human leukocyte antigen (HLA))Class I or MHC Class II molecule, are recognized and bound by a T-cellreceptor (TCR) present on a T cell surface. T-cell epitopes includeepitopes recognized by cytotoxic T cells (e.g., CD8⁺ T cells), andepitopes recognized by helper T cells (e.g., CD4⁺ T cells).

The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) anHCV NS4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NSSApolypeptide; f) an HCV NSSB polypeptide; g) an HCV core polypeptide; orh) an HCV p7 polypeptide. In some cases, the one or more T-cell epitopesare T-cell epitopes present in an HCV NS3 polypeptide. In some cases,the heterologous polypeptide further comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) ameningococcal outer membrane protein. Other examples of strong T helperepitopes are diphtheria toxoid, tetanus toxoid, meningococcal outermembrane protein, or mutant diphtheria protein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127).

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS3 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS3 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS3 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS3 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS3 CD4⁺ T cell epitope and at least one HCV-NS3 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS3 CD4⁺ T-cell epitopes and 2 or more HCV-NS3 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS3 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS3 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS2 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS2 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS2 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS2 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS2 CD4⁺ T cell epitope and at least one HCV-NS2 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS2 CD4⁺ T-cell epitopes and 2 or more HCV-NS2 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS2 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS2 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS4A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS4A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS4A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS4A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS4A CD4⁺ T cell epitope and at least one HCV-NS4A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS4A CD4⁺ T-cell epitopes and 2 or more HCV-NS4A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS4A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS4A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5A CD4⁺ T cell epitope and at least one HCV-NS5A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5A CD4⁺ T-cell epitopes and 2 or more HCV-NS5A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5B T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5B T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5B T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5B T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5B CD4⁺ T cell epitope and at least one HCV-NS5B CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5B CD4⁺ T-cell epitopes and 2 or more HCV-NS5B CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5B CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5B CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-core T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-core T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-core T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-core CD4⁺ T cell epitope and at least one HCV-core CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-core CD4⁺ T-cell epitopes and 2 or more HCV-core CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-core CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-core CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-p7 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-p7 T-cell epitopes.In some cases, the heterologous polypeptide comprises 4 or more HCV-p7T-cell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD4⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core CD4⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises one or more HCV CD8⁺ Tcell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD8⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 CD8⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope. In some cases,the heterologous polypeptide comprises at least one HCV-p7 CD4⁺ T cellepitope and at least one HCV-p7 CD8⁺ T cell epitope. In some cases,heterologous polypeptide comprises 2 or more HCV-p7 CD4⁺ T-cell epitopesand 2 or more HCV-p7 CD8⁺ T-cell epitopes. In some cases, theheterologous polypeptide comprises 2, 3, 4, or 5 HCV-p7 CD4⁺ T-cellepitopes and 2, 3, 4, or 5 HCV-p7 CD8⁺ T-cell epitopes.

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, or 63, of the T-cell epitopes set out in FIG. 13A-13B. In somecases, the heterologous polypeptide comprises from 1 to 3, from 3 to 5,from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, or from 25 to30 of the T-cell epitopes set out in FIG. 13A-13B. For example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS3-3, NS3-4, and NS3-11 in FIG. 13A-13B and FIG. 15A-15N. Asanother example, in some cases, the heterologous polypeptide comprisesthe T-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 inFIG. 13A-13B and FIG. 15A-15N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-3,NS3-4, NS3-5, and NS3-11 in FIG. 13A-13B and FIG. 15A-15N. As anotherexample, in some cases, the heterologous polypeptide comprises theT-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated Core-1, Core-2, Core-3, Core-4, Core-5, Core-6,Core-7, Core-8, Core-9, Core-10, Core-11, Core-12, Core-13, Core-14,Core-16, Core-17, Core-18, Core-19, Core-20, Core-21, and Core-22 inFIG. 13A-13B and FIG. 15A-15N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-1,NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6, NS2-7, NS2-8,NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1,NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, andNS4b-10 in FIG. 13A-13B and FIG. 15A-15N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8,NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2,NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1,NS5a-2, NS5b-1, and NS5b-2 in FIG. 13A-13B and FIG. 15A-15N. In somecases, the T-cell epitopes are contiguous. In some cases, any two T-cellepitopes are separated by linkers (e.g., a linker having a length offrom 1 amino acid to about 50 amino acids, e.g., from 1 amino acid to 5amino acids (aa), from 5 aa to 10 aa, from 10 aa to 15 aa, from 15 aa to20 aa, from 20 aa to 25 aa, from 25 aa to 30 aa, from 30 aa to 40 aa, orfrom 40 aa to 50 aa).

In some cases, the heterologous polypeptide comprises at least one HCVCD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope, whereepitopes are conserved among HCV genotypes 1 and 2. In some cases, theheterologous polypeptide comprises at least one HCV CD4⁺ T cell epitopeand at least one HCV CD8⁺ T cell epitope, where epitopes are conservedamong HCV genotypes 1 and 3. In some cases, the heterologous polypeptidecomprises at least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺T cell epitope, where epitopes are conserved among HCV genotypes 1, 2,and 3. In some cases, the heterologous polypeptide comprises at leastone HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope,where epitopes are conserved among HCV genotypes 1, 2, 3, and 7. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope, where epitopesare conserved among HCV genotypes 1-7.

The heterologous polypeptide can have a length of from about 10 aminoacids to about 2000 amino acids; e.g., the heterologous polypeptide canhave a length of from 10 amino acids (aa) to 15 aa, from 15 aa to 20 aa,from 20 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to 75 aa, from 75aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aato 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to400 aa, from 450 aa to 500 aa, from 500 aa to 550 aa, from 550 aa to 600aa, from 600 aa to 650 aa, from 650 aa to 700 aa, from 700 aa to 750 aa,or from 750 aa to 800 aa. The heterologous polypeptide can have a lengthof from about 25 amino acids to about 2000 amino acids, e.g., from about25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa,from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aa to 250 aa,from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to 400 aa,from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to 700 aa,from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to 1000 aa,from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aa to 1300aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500 aa to1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from 1800 aato 1900 aa, or from 1900 aa to 2000 aa. The heterologous polypeptide canhave a length of from about 25 amino acids to about 3000 amino acids,e.g., from about 25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aato 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to700 aa, from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to1000 aa, from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aato 1300 aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500aa to 1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from1800 aa to 1900 aa, from 1900 aa to 2000 aa, from 2000 aa to 2250 aa,from 2250 aa to 2500 aa, from 2500 aa to 2750 aa, or from 2750 aa to3000 aa.

The heterologous polypeptide can have a length of from about 25 aminoacids to about 800 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from about 25 aminoacids to about 400 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, or from 350 aa to 400 aa. The heterologouspolypeptide can have a length of 25 amino acids (aa), 26 aa, 27 aa, 28aa, 29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa, 36 aa, 37 aa, 38aa, 39 aa, 40 aa, 41 aa, 42 aa, 43 aa, 44 aa, 45 aa, 46 aa, 47 aa, 48aa, 49 aa, or 50 aa. The heterologous polypeptide can have a length offrom about 100 amino acids (aa) to 800 aa, e.g., from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from 25 aa to 30 aa.The heterologous polypeptide can have a length of from 30 aa to 40 aa.The heterologous polypeptide can have a length of from 40 aa to 50 aa.The heterologous polypeptide can have a length of from 50 aa to 60 aa(e.g., 50 aa, 51 aa, 52, aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa,59 aa, or 60 aa). The heterologous polypeptide can have a length of from60 aa to 70 aa. The heterologous polypeptide can have a length of from65 aa to 75 aa (e.g., 65, 66, 67, 68, 69, 70, 71, 72, 7, 74, or 75 aa).The heterologous polypeptide can have a length of 70 aa. Theheterologous polypeptide can have a length of from 70 aa to 80 aa. Theheterologous polypeptide can have a length of from 80 aa to 90 aa. Theheterologous polypeptide can have a length of from 90 aa to 100 aa. Theheterologous polypeptide can have a length of from 100 aa to 105 aa(e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 aa). Theheterologous polypeptide can have a length of 100 aa. The heterologouspolypeptide can have a length of from 10 amino acids (aa) to 50 aa;e.g., from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to 25 aa,from 25 aa to 30 aa, from 30 aa to 35 aa, from 35 aa to 40 aa, from 40aa to 45 aa, or from 45 aa to 50 aa. The heterologous polypeptide canhave a length of from 10 amino acids (aa) to 20 aa, e.g., 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 aa.

HCV NS3 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS3 polypeptide. Examplesof T-cell epitopes present in NS3 polypeptides are depicted in FIG.15A-15N, FIG. 13B, and FIG. 14A-14B.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1). In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1); and has a length of from 25aa to 35 aa (e.g., 25 aa, 26 aa, 27 aa, 28 aa, 29 aa, 30 aa, 31 aa, 32aa, 33 aa, 34 aa, or 35 aa). In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1); and has alength of 29 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-3, NS3-4, and NS3-11 in FIG. 13B and FIG.15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2); andhas a length of from 45 amino acids to 60 amino acids (e.g., 45 aa, 46aa, 47 aa, 48 aa, 49 aa, 50 aa, 51 aa, 52 aa, 53 aa, 54 aa, 55 aa, 56aa, 57 aa, 58 aa, 59 aa, or 60 aa). In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2); andhas a length of 52 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-3, NS3-4, NS3-5, and NS3-11 in FIG. 13B and FIG.15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:3); and has a length of from 65 amino acids to 80 amino acids(e.g., 65 aa, 66 aa, 67 aa, 68 aa, 69 aa, 70 aa, 71 aa, 72 aa, 73 aa, 74aa, 75 aa, 76 aa, 77 aa, 78 aa, 79 aa, or 80 aa). In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:3); and has a length of 70 amino acids. Such a polytope caninclude NS3 T-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6,NS3-7, NS3-11, NS3-12, and NS3-13 in FIG. 13B and FIG. 15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4); and has a lengthof from 95 amino acids (aa) to 105 aa (e.g., 95 aa, 96 aa, 97 aa, 98 aa,99 aa, 100 aa, 101 aa, 102 aa, 103 aa, 104 aa, or 105 aa). In somecases, the heterologous polypeptide comprises an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4); and has a lengthof 100 amino acids. Such a polytope can include NS3 T-cell epitopesdesignated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 13B and FIG. 15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:9); and has a length of from 190 aminoacids (aa) to 200 aa (e.g., 190 aa, 191 aa, 192 aa, 193 aa, 194 aa, 195aa, 196 aa, 197 aa, 198 aa, 199 aa, or 200 aa. In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:9); and has a length of 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:10); and hasa length of from 215 amino acids (aa) to 235 aa (e.g., 215 aa, 216 aa,217 11, 218 aa, 219 aa, 220 aa, 221 aa, 222 aa, 223 aa, 224 aa, 225 aa,226 aa, 227 aa, 228 aa, 229 aa, 230 aa, 231 aa, 232 aa, 233 aa, 234 aa,or 235 aa). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:10); and hasa length of 228 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 13B and FIG. 15A-15N.

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1265-1279 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1309-1323 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1401-1415 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1402-1412 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1429-1439 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1464 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1453-1467 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1577-1591 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1306-1314 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1387-1394 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 1amino acids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12aa, 13 aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1405-1413 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1458 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1457-1465 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1610-1618 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

HCV NS2 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS2 polypeptide. Examplesof T-cell epitopes present in NS2 polypeptides are depicted in FIG.15A-15N, and FIG. 13A.

For example, the heterologous polypeptide can comprise an NS2 T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids955-974 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 975-994 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 985-1004 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1015-1034 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1035-1054 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 924-933 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 961-970 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 989-997 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 50 aa (e.g., from 10 aa to 25 aa, or from 25 aa to 50 aa) ofamino acids 955-1004 of the amino acid sequence designated “Consensus”in FIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa, orfrom 25 aa to 50 aa. In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 955-1004of the amino acid sequence designated “Consensus” in FIG. 16A-16L, or acorresponding HCV NS2 amino acid sequence of any HCV genotype; and has alength of about 50 amino acids.

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 553 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from 300 aato 400 aa, from 400 aa to 500 aa, or from 500 aa to 553 aa) of aminoacids 917-1469 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 and NS3 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa,from 25 aa to 50 aa, from 50 aa to 100 aa, from 100 aa to 200 aa, from200 aa to 300 aa, from 300 aa to 400 aa, from 400 aa to 500 aa, or from500 aa to 553 aa. In some cases, the heterologous polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 917-1469 of the aminoacid sequence designated “Consensus” in FIG. 16A-16L, or a correspondingHCV NS2 and NS3 amino acid sequence of any HCV genotype; and has alength of about 553 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 0%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11); andhas a length of from 50 amino acids to 60 amino acids (e.g., 50 aa, 51aa, 52 aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60 aa).In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11); andhas a length of 50 amino acids. Such a polytope can include NS2 T-cellepitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 in FIG. 13Aand FIG. 15A-15N.

HCV NS4A T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS4A polypeptide. Examplesof T-cell epitopes present in NS4A polypeptides are depicted in FIG.15A-15N and FIG. 13B.

The heterologous polypeptide can comprise an NS4A T cell epitopecomprising an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids1683-1692 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS4A amino acid sequence of any HCVgenotype; and the NS4A T-cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

HCV NS4B T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS4B polypeptide. Examplesof T-cell epitopes present in NS4B polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NS4B T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids1790-1801 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 12 aminoacids (aa) to 20 amino acids (e.g., 12 aa, 13 aa, 14 aa, 15 aa, 16 aa,17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1792-1802 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 11 aminoacids (aa) to 20 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15 aa,16 aa, 17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1898-1905 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 8 aminoacids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1921-1935 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1922-1941 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1928-1947 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1868-1876 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1927-1942 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 16 aminoacids (aa) to 20 amino acids (e.g., 16 aa, 17 aa, 18 aa, 19 aa, or 20aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1932-1940 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1948-1962 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

HCV NS5A T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS5A polypeptide. Examplesof T-cell epitopes present in NS5A polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NS5A T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2218-2232 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NSSA amino acid sequence of any HCVgenotype; and the NSSA T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NSSA Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2309-2317 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NSSA amino acid sequence of any HCVgenotype; and the NSSA T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV NSSB T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NSSB polypeptide. Examplesof T-cell epitopes present in NSSB polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NS5B T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2847-2851 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS5B amino acid sequence of any HCVgenotype; and the NS5B T-cell epitope can have a length of from 5 aminoacids (aa) to 10 amino acids (e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10aa).

As another example, the heterologous polypeptide can comprise an NS5B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2602-2610 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS5B amino acid sequence of any HCVgenotype; and the NS5B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV Core T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV core polypeptide. Examplesof T-cell epitopes present in HCV Core polypeptides are depicted in FIG.15A-15N and FIG. 13A.

As one example, the heterologous polypeptide can comprise an HCV core Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1-20 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 11-30 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 21-40 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 39-63 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 23amino acids (aa) to 28 amino acids (e.g., 23 aa, 24 aa, 25 aa, 26 aa, 27aa, or 28 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 47-70 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 24amino acids (aa) to 29 amino acids (e.g., 24 aa, 25 aa, 26 aa, 27 aa, 28aa, or 29 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 61-80 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 71-90 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 81-100 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 91-110 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 101-115 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 111-130 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 125-139 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-150 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 151-170 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 161-180 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 35-44 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 43-51 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 51-59 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 129-137 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-140 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 150-158 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 154-162 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 168-176 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 177-187 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 178-187 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 191 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, or from 150 aato 191 aa) of amino acids 1-191 of the amino acid sequence designated“Consensus” in FIG. 16A-16L, or a corresponding HCV core amino acidsequence of any HCV genotype; and has a length of from 10 amino acids(aa) to 25 aa, from 25 aa to 50 aa, from 50 aa to 100 aa, or from 100 aato 150 aa, or from 150 aa to 191 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to amino acids1-191 of the amino acid sequence designated “Consensus” in FIG. 16A-16L,or a corresponding HCV core amino acid sequence of any HCV genotype; andhas a length of about 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLE DGVNYATGNLPG (SEQID NO:63); and has a length of from 171 amino acids (aa) to 180 aa(e.g., 171 aa, 172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa,179 aa, or 180 aa. In some cases, the heterologous polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLE DGVNYATGNLPG (SEQID NO:63); and has a length of 171 amino acids. Such a polytope caninclude core T-cell epitopes designated Core-1, Core-2, Core-3, Core-4,Core-5, Core-6, Core-7, Core-8, Core-9, Core-10, Core-11, Core-12,Core-13, Core-14, Core-16, Core-17, Core-18, Core-19, Core-20, Core-21,Core-22 in FIG. 13A and FIG. 15A-15N.

HCV p7 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV p7 polypeptide. Examplesof T-cell epitopes present in HCV p7 polypeptides are depicted in FIG.15A-15N or FIG. 13A.

As another example, the heterologous polypeptide can comprise an HCV p7T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 803-811 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV p7 amino acid sequence of any HCVgenotype; and the HCV p7 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

Polytopes Including HCV T-Cell Epitopes from More than One HCVPolypeptide Other than E1 and E2

As noted above, a heterologous polypeptide can include T-cell epitopesfrom more than one HCV polypeptide other than E1 and E2.

As one example, a heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTG DFDSVIDCN (SEQ IDNO:12); and has a length of from 550 amino acids (aa) to 560 aa (e.g.,550 aa, 551 aa, 552 aa, 553 aa, 554 aa, 555 aa, 556 aa, 557 aa, 558 aa,559 aa, or 560 aa). In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTG DFDSVIDCN (SEQ IDNO:12); and has a length of 553 amino acids. Such a polytope can includeT-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6,NS2-7, NS2-8, NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9,NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N.This polytope is also referred to as “TP553” (FIG. 14A-14D). In order toprevent self cleavage of the TP553 polytope (amino acids 917-1469) (FIG.15E-G) at the NS2-NS3 junction that is mediated by the catalytic domainof the NS2 protease (amino acids 917-1040), the histidine at position966 (H966), a critical residue for NS2 protease activity, is mutated toalanine (H966A) (FIG. 15E).

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aato 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa) the followingamino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to a contiguous stretch of from 25amino acids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa,from 200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa,from 350 aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa)of the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64); and has a length of from 25 amino acids (aa) to 50 aa,from 50 aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from300 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600aa to 700 aa, or from 700 aa to 778 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64); and has a length of 778 amino acids. Such a polytope caninclude T-cell epitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5,NS3-6, NS3-7, NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS2-14,NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8,NS4b-9, and NS4b-10 in FIG. 13B and FIG. 15A-15N.

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 1985 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 500 aa, from 500 aa to 750 aa, from 750aa to 1000 aa, from 1000 aa to 1500 aa, or from 1500 aa to 1985 aa) ofthe following amino acid sequence:

(SEQ ID NO: 13) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTV YHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVT RHADVIPVRRRGDSRGSLLSPRPISYLKGS A GGPLLCPAGHAVGIFRAAVCTRGV AKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAA YAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYG KFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPP GSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLV ALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQT VDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSV LCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHID AHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPT PLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCL STGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQ KALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPG NPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGA AIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGAL VVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARV TAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKL MPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTC RNMWSGTFPINTAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGM TTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQL PCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCT ANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVP AEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPP RKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAE SYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAE EQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEV KAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVW KDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYD VVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTE SDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVL TTSCGNTLTCYIKARAACRAAGLQDCTMLVCG NN LVVICESAGVQEDAASLRA FTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLA RAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEI YGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHR ARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR. 

In some cases, the heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGMTTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPPRKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGNNLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR (SEQ ID NO:13); and has a length of1985 amino acids. Such a polytope can include T-cell epitopes designatedNS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8, NS3-9, NS3-10,NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4,NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1, NS5a-2, NS5b-1,NS5b-2 in FIG. 13A-13B and FIG. 15A-15N.

Additional T-Cell Epitopes

As discussed above, an E1/E2 a heterodimeric polypeptide of the presentdisclosure includes: a) an HCV E2 polypeptide; and b) a variant HCV E1polypeptide comprising: i) an HCV E1 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide). The one or more T-cell epitopescan include one or more T-cell epitopes present in: a) an HCV NS3polypeptide; b) an HCV NS2 polypeptide; c) an HCV NS4A polypeptide; d)an HCV NS4B polypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5Bpolypeptide; g) an HCV core polypeptide; or h) an HCV p7 polypeptide. Insome cases, the one or more T-cell epitopes are T-cell epitopes presentin an HCV NS3 polypeptide. In some cases, the heterologous polypeptidefurther comprises one or more T cell epitopes present in: a) choleratoxin or toxoid; and/or b) tetanus toxin or toxoid; and/or c) diphtheriatoxin or toxoid; and/or d) a meningococcal outer membrane protein.

Thus, in some cases, a variant HCV E1 polypeptide of an E1/E2heterodimer of the present disclosure includes: a) an HCV E1polypeptide; and b) a heterologous polypeptide that comprises one ormore T-cell epitopes, where the one or more T-cell epitopes are T-cellepitopes present in: i) one or more of an HCV NS3 polypeptide, an HCVNS2 polypeptide, an HCV NS4A polypeptide, an HCV NS4B polypeptide, anHCV NS5A polypeptide, an HCV NS5B polypeptide, an HCV core polypeptide,and an HCV p7 polypeptide; and ii) one or more of cholera toxin ortoxoid, tetanus toxin or toxoid, diphtheria toxin or toxoid, and ameningococcal outer membrane protein.

Additional T-Cell Epitopes

As discussed above, an E1/E2 a heterodimeric polypeptide of the presentdisclosure includes: a) an HCV E1 polypeptide; and b) a variant HCV E2polypeptide comprising: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide). The one or more T-cell epitopescan include one or more T-cell epitopes present in: a) an HCV NS3polypeptide; b) an HCV NS2 polypeptide; c) an HCV NS4A polypeptide; d)an HCV NS4B polypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5Bpolypeptide; g) an HCV core polypeptide; or h) an HCV p7 polypeptide. Insome cases, the one or more T-cell epitopes are T-cell epitopes presentin an HCV NS3 polypeptide. In some cases, the heterologous polypeptidefurther comprises one or more T cell epitopes present in: a) choleratoxin or toxoid; and/or b) tetanus toxin or toxoid; and/or c) diphtheriatoxin or toxoid; and/or d) a meningococcal outer membrane protein.

Thus, in some cases, a variant HCV polypeptide of an E1/E2 heterodimerof the present disclosure includes: a) an HCV E2 polypeptide; and b) aheterologous polypeptide that comprises one or more T-cell epitopes,where the one or more T-cell epitopes are T-cell epitopes present in: i)one or more of an HCV NS3 polypeptide, an HCV NS2 polypeptide, an HCVNS4A polypeptide, an HCV NS4B polypeptide, an HCV NS5A polypeptide, anHCV NS5B polypeptide, an HCV core polypeptide, and an HCV p7polypeptide; and ii) one or more of cholera toxin or toxoid, tetanustoxin or toxoid, diphtheria toxin or toxoid, and a meningococcal outermembrane protein.

A T helper tetanus toxin epitope or other bacterial T-cell epitope couldbe fused (e.g., by recombinant expression) or chemically conjugated tothe HCV polytope/E2 fusion protein and/or to the HCV polytope E1 fusionprotein of an E/E2 heterodimer of the present disclosure to furtherenhance both T and B cell responses to both the HCV polytope and E1/E2moieties. Alternatively, the whole or part of the detoxified toxin(“toxoid”) could be fused (e.g., by recombinant expression) orchemically conjugated to the HCV polytope/E1E2 protein, wherein specificamino acids of the toxins are mutated to render the toxins inactive,thereby generating toxoids. Methods of generating toxoids are well knownin the art. Examples of bacterial epitopes include the use of diphtheriatoxoid, meningococcal outer membrane protein, or mutant diphtheriaprotein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127)

In some cases, a suitable tetanus toxoid polypeptide comprises the aminoacid sequence QYIKANSKFIGIFE (SEQ ID NO:14). In some cases, a suitabletetanus toxoid polypeptide comprises the amino acid sequenceQYIKANSKFIGITE (SEQ ID NO:65).

In some cases, a heterologous polypeptide can comprise cholera toxin (ortoxoid) epitope. In some cases, a suitable heterologous polypeptidecomprising a cholera toxoid epitope comprises a fragment of choleratoxin-B subunit (CT-B), e.g., a fragment of from 5 amino acids to 25amino acids, or from 25 amino acids to 50 amino acids, of the followingamino acid sequence: MIKLKFGVFF TVLLSSAYAH GTPQNITDLC AEYHNTQIHTLNDKIFSYTE SLAGKREMAI ITFKNGATFQ VEVPGSQHID SQKKAIERMK DTLRIAYLTEAKVEKLCVWN NKTPHAIAAI SMAN (SEQ ID NO:15).

In some cases, a heterologous polypeptide can comprise a tetanus toxin(or toxoid) T-cell epitope. In some cases, a suitable heterologouspolypeptide comprising a tetanus toxin T-cell epitope comprises theamino acid sequence: ILMQYIKANSKFIGI (SEQ ID NO:16); and has a length offrom 15 amino acids to 20 amino acids. In some cases, a suitableheterologous polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: VNNESSE (SEQ ID NO:17). In somecases, a suitable heterologous polypeptide comprising a tetanus toxinT-cell epitope comprises the amino acid sequence: PGINGKAIHLVNNESSE (SEQID NO:18). In some cases, a suitable heterologous polypeptide comprisinga tetanus toxin T-cell epitope comprises the amino acid sequence: PNRDIL(SEQ ID NO:19). In some cases, a suitable heterologous polypeptidecomprising a tetanus toxin T-cell epitope comprises the amino acidsequence: FIGITEL (SEQ ID NO:20). In some cases, a suitable tetanustoxin T-cell epitope comprises the amino acid sequence: SYFPSV (SEQ IDNO:21). In some cases, a suitable heterologous polypeptide comprising atetanus toxin T-cell epitope comprises the amino acid sequence:NSVDDALINSTKIYSYFPSV (SEQ ID NO:22). In some cases, a suitableheterologous polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: IDKISDVSTIVPYIGPALNI (SEQ ID NO:23).

In some cases, a heterologous polypeptide can comprise a diphtheriatoxin T-cell epitope In some cases, a suitable heterologous polypeptidecomprising a diphtheria toxin T-cell epitope comprises the amino acidsequence: QSIALSSLMVAQAIP (SEQ ID NO:24); and has a length of from 15amino acids to 20 amino acids. In some cases, a suitable heterologouspolypeptide comprising a diphtheria toxin T-cell epitope comprises theamino acid sequence: PVFAGANYAAWAVNVAQVI (SEQ ID NO:25). In some cases,a suitable heterologous polypeptide comprising a diphtheria toxin T-cellepitope comprises the amino acid sequence: VHHNTEEIVAQSIALSSLMV (SEQ IDNO:26). In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QSIALSSLMVAQAIPLVGEL (SEQ ID NO:66). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: VDIGFAAYNFVESIINLFQV (SEQ ID NO:67).In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QGESGHDIKITAENTPLPIA (SEQ ID NO:68). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: GVLLPTIPGKLDVNKSKTHI (SEQ ID NO:69).In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence ofCRM197 (see, e.g., Giannini et al. (1984) Nucl. Acids. Res. 12:4063).The amino acid sequence of CRM197 is provided above.

In some cases, a heterologous polypeptide can comprise a tetanus toxinT-cell epitope and a diphtheria toxin T-cell epitope. In some of thesecases, the heterologous polypeptide can comprise the amino acidsequence: IMQYIKANSKFIGIQSIALSSLMVAQ (SEQ ID NO:28); and can have alength of from 26 amino acids to 30 amino acids.

E2

An E2 polypeptide suitable for inclusion in an E1/E2 heterodimer of thepresent disclosure can have a length of from about 200 amino acids (aa)to about 250 aa, from about 250 aa to about 275 aa, from about 275 aa toabout 300 aa, from about 300 aa to about 325 aa, from about 325 aa toabout 350 aa, or from about 350 aa to about 365 aa. In some cases, an E2polypeptide suitable for inclusion in an E1/E2 heterodimer of thepresent disclosure is a full-length HCV E2 polypeptide. In some cases,an E2 polypeptide suitable for inclusion in an E1/E2 heterodimer of thepresent disclosure is an HCV E2 ectodomain polypeptide.

In FIG. 1A-AC, the amino acid sequence of E2 is amino acid 384 to aminoacid 746. In FIG. 2A-2B, the amino acid sequence of E2 is amino acid 384to amino acid 751. In FIG. 3A-3C, the amino acid sequence of E2 is aminoacid 385 to amino acid 754. In FIG. 4A-4B, the amino acid sequence of E2is amino acid 384 to amino acid 750. As used herein, an “E2 polypeptide”includes a precursor E2 protein, including the signal sequence; includesa mature E2 polypeptide which lacks this sequence; and includes an E2polypeptide with a heterologous signal sequence. An E2 polypeptide caninclude a C-terminal membrane anchor sequence which occurs atapproximately amino acid positions 715-730 and may extend as far asapproximately amino acid residue 746 (see, Lin et al., J. Virol. (1994)68:5063-5073).

In some cases, a E2 polypeptide suitable for inclusion in an E1/E2heterodimer of the present disclosure lacks a portion of its C-terminalregion, e.g., from about amino acid 715 to the C-terminus; from aboutamino acid 625 to the C-terminus; from about amino acid 661 to theC-terminus; from about amino acid 655 to the C-terminus; from aboutamino acid 500 to the C-terminus, where the amino acid numbering is withreference to the numbering in FIG. 1A-1C. See, e.g., U.S. Pat. No.6,521,423.

An E2 polypeptide suitable for inclusion in an E1/E2 heterodimer of thepresent disclosure can comprise an amino acid sequence having having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG. 4A-4B.

An E2 polypeptide suitable for inclusion in an E1/E2 heterodimer of thepresent disclosure can comprise an amino acid sequence having having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.1A-1C. For example, an E2 polypeptide of genotype 1 can comprise anamino acid sequence having having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 384-746 of anamino acid sequence depicted in FIG. 1A-1C. For example, an E2polypeptide of genotype 1A can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 384-746 of an amino acid sequenceidentified as 1A and depicted in FIG. 1A-1C. For example, an E2polypeptide of genotype 1B can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 384-746 of an amino acid sequenceidentified as 1B and depicted in FIG. 1A-1C. For example, an E2polypeptide of genotype 1C can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 384-746 of an amino acid sequenceidentified as 1C and depicted in FIG. 1A-1C.

An E2 polypeptide suitable for inclusion in an E1/E2 heterodimer of thepresent disclosure can comprise an amino acid sequence having having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.2A-2C. For example, an E2 polypeptide can comprise an amino acidsequence having having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to amino acids 384-746 of an amino acidsequence depicted in FIG. 2A-2C. For example, an E2 polypeptide ofgenotype 2A can comprise an amino acid sequence having having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 384-751 of the “consensus” amino acid sequence depicted inFIG. 2A-2C. For example, an E2 polypeptide of genotype 2B can comprisean amino acid sequence having having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 384-751 of the“consensus” amino acid sequence depicted in FIG. 2A-2C.

An E2 polypeptide suitable for inclusion in an E1/E2 heterodimer of thepresent disclosure can comprise an amino acid sequence having having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.3A-3C. For example, an E2 polypeptide of genotype 3 can comprise anamino acid sequence having having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 385-754 of anamino acid sequence depicted in FIG. 3A-3C. For example, an E2polypeptide of genotype 3A can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 385-754 of an amino acid sequenceidentified as 3A and depicted in FIG. 3A-3C. For example, an E2polypeptide of genotype 3B can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 385-754 of the amino acid sequenceidentified as 3B and depicted in FIG. 3A-3C. For example, an E2polypeptide of genotype 3K can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 385-754 of the amino acid sequenceidentified as 3K and depicted in FIG. 3A-3C.

An E2 polypeptide suitable for inclusion in an E1/E2 heterodimer of thepresent disclosure can comprise an amino acid sequence having having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the amino acid sequence of the E2 polypeptide depicted inFIG. 4A-4B. For example, an E2 polypeptide of genotype 7A can comprisean amino acid sequence having having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 384-750 of theamino acid sequence depicted in FIG. 4A-4B.

Additional Polypeptides

In any of the above-described embodiments, one or both of thepolypeptide chains of the E1/E2 heterodimer can include one or moreadditional polypeptides. For example, in some cases, the variant E1polypeptide or the E2 polypeptide can include an Ig Fc polypeptide atthe C-terminus of the variant E1 polypeptide or the E2 polypeptide. Asanother example, in some cases, the variant E1 polypeptide or the E2polypeptide can include an Ig Fc polypeptide at the N-terminus of thevariant E1 polypeptide or the E2 polypeptide. Ig Fc polypeptides areknown in the art, and are described elsewhere herein.

IC. E1E2 Heterodimers Comprising a Variant HCV E1 and a Variant HCV E2

The present disclosure provides an E1/E2 heterodimer, where the E1/E2heterodimer includes both a variant E1 polypeptide and a variant E2polypeptide.

Thus, in some cases, a heterodimeric polypeptide of the presentdisclosure includes: a) a variant HCV E1 polypeptide comprising: i) anHCV E1 polypeptide; and ii) a heterologous polypeptide that comprisesone or more T-cell epitopes (e.g., one or more T cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide); and b) a variant HCV E2 polypeptide comprising: i) an HCVE2 polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). The one or more T-cell epitopes can include one or moreT-cell epitopes present in: a) an HCV NS3 polypeptide; b) an HCV NS2polypeptide; c) an HCV NS4A polypeptide; d) an HCV NS4B polypeptide; e)an HCV NS5A polypeptide; f) an HCV NS5B polypeptide; g) an HCV corepolypeptide; or h) an HCV p7 polypeptide. In some cases, the one or moreT-cell epitopes are T-cell epitopes present in an HCV NS3 polypeptide.In some cases, the heterologous polypeptide further comprises one ormore T cell epitopes present in: a) cholera toxin or toxoid; and/or b)tetanus toxin or toxoid; and/or c) diphtheria toxin or toxoid; and/or d)a meningococcal outer membrane protein. In some cases, a variant HCV E2and a variant HCV E1 polypeptide of an E1/E2 heterodimer of the presentdisclosure includes: 1) a) an HCV E2 polypeptide; and b) a heterologouspolypeptide that comprises one or more T-cell epitopes, where the one ormore T-cell epitopes are T-cell epitopes present in: i) one or more ofan HCV NS3 polypeptide, an HCV NS2 polypeptide, an HCV NS4A polypeptide,an HCV NS4B polypeptide, an HCV NS5A polypeptide, an HCV NS5Bpolypeptide, an HCV core polypeptide, and an HCV p7 polypeptide; and ii)one or more of cholera toxin or toxoid, tetanus toxin or toxoid,diphtheria toxin or toxoid, and a meningococcal outer membrane protein;and 2) a) an HCV E1 polypeptide; and b) a heterologous polypeptide thatcomprises one or more T-cell epitopes, where the one or more T-cellepitopes are T-cell epitopes present in: i) one or more of an HCV NS3polypeptide, an HCV NS2 polypeptide, an HCV NS4A polypeptide, an HCVNS4B polypeptide, an HCV NS5A polypeptide, an HCV NS5B polypeptide, anHCV core polypeptide, and an HCV p7 polypeptide; and ii) one or more ofcholera toxin or toxoid, tetanus toxin or toxoid, diphtheria toxin ortoxoid, and a meningococcal outer membrane protein.

In some cases, a variant E2 polypeptide present in an E1/E2 heterodimerof the present disclosure comprises, in order from N-terminus toC-terminus: i) an HCV E2 polypeptide; and ii) a heterologous polypeptidecomprising one or more T cell epitopes. In some cases, a variant HCV E2polypeptide present in an E1/E2 heterodimer of the present disclosurecomprises, in order from N-terminus to C-terminus: i) a heterologouspolypeptide comprising one or more T cell epitopes; and ii) an HCV E2polypeptide. In some cases, a variant E1 polypeptide present in an E1/E2heterodimer of the present disclosure comprises, in order fromN-terminus to C-terminus: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide comprising one or more T cell epitopes. In somecases, a variant HCV E1 polypeptide present in an E1/E2 heterodimer ofthe present disclosure comprises, in order from N-terminus toC-terminus: i) a heterologous polypeptide comprising one or more T cellepitopes; and ii) an HCV E1 polypeptide. In some cases, both the variantE1 and the variant E2 polypeptide include the heterologous polypeptideat the N-terminus of the E1 and E2 polypeptides. In some cases, both thevariant E1 and the variant E2 polypeptide include the heterologouspolypeptide at the C-terminus of the E1 and E2 polypeptides. In somecases, the variant E1 polypeptide comprises the heterologous polypeptideat the N-terminus of the E1 polypeptide; and the variant E2 polypeptidecomprises the heterologous polypeptide at the C-terminus of the E2polypeptide. In some cases, the variant E1 polypeptide comprises theheterologous polypeptide at the C-terminus of the E1 polypeptide; andthe variant E2 polypeptide comprises the heterologous polypeptide at theN-terminus of the E2 polypeptide.

Variant E1

As noted above, a variant E1 polypeptide of an HCV E1/E2 heterodimer ofthe present disclosure comprises: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide comprising one or more T cell epitopes (e.g.,one or more T-cell epitopes not present in an HCV E1 or an HCV E2polypeptide; e.g., one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 or an HCV E2 polypeptide). In somecases, the variant HCV E1 polypeptide comprises, in order from aminoterminus (N-terminus) to carboxyl terminus (C-terminus): i) an HCV E1polypeptide; and ii) a heterologous polypeptide comprising one or more Tcell epitopes. In some cases, the variant HCV E1 polypeptide comprises,in order from N-terminus to C-terminus: i) a heterologous polypeptidecomprising one or more T cell epitopes; and ii) an HCV E1 polypeptide.

E1

An HCV E1 polypeptide suitable for inclusion in a variant E1 polypeptideof an E1/E2 heterodimer of the present disclosure can have a length offrom about 150 amino acids (aa) to about 175 aa, from about 175 aa toabout 195 aa, from about 131 aa to about 175 aa, or from about 175 aa toabout 193 aa. In some cases, an E1 polypeptide suitable for inclusion ina variant E1 polypeptide of an HCV E1/E2 heterodimer of the presentdisclosure is a full-length HCV E1 polypeptide. In some cases, an E1polypeptide suitable for inclusion in a variant E1 polypeptide of an HCVE1/E2 heterodimer of the present disclosure is an HCV E1 ectodomainpolypeptide.

In FIG. 1A-1C, the amino acid sequence of E1 is amino acid 192 to aminoacid 383. In FIG. 2A-2C, the amino acid sequence of E1 is amino acid 192to amino acid 383. In FIG. 3A-3C, the amino acid sequence of E1 is aminoacid 192 to amino acid 384. In FIG. 4A-4B, the amino acid sequence of E1is amino acid 192 to amino acid 383. Amino acids at around 170 throughapproximately 191 serve as a signal sequence for E1. As used herein, “E1polypeptide” includes a precursor E1 protein, including the signalsequence; includes a mature E1 polypeptide which lacks this sequence;and includes an E1 polypeptide with a heterologous signal sequence. AnE1 polypeptide can include a C-terminal membrane anchor sequence whichoccurs at approximately amino acid positions 360-383 (see, e.g., WO96/04301). In some cases, a suitable E1 polypeptide lacks a C-terminalportion that includes a transmembrane region. For example, in somecases, a suitable E1 polypeptide lacks the C-terminal portion from aminoacid 330 to amino acid 384, or from amino acid 360 to amino acid 384. E1polypeptides can be an E1 polypeptide of any genotype, subtype orisolate of HCV. E1 polypeptides of genotype 1 and E1 polypeptides ofgenotype 3 are included in an E1/E2 heterodimer of the presentdisclosure.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence of an E1polypeptide depicted in FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG.4A-4B.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence of an E1polypeptide depicted in FIG. 1A-1C. For example, an E1 polypeptide ofgenotype 1A can comprise an amino acid sequence having having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 192-383 of an amino acid sequence identified as 1A anddepicted in FIG. 1A-1C. For example, an E1 polypeptide of genotype 1Bcan comprise an amino acid sequence having having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids192-383 of an amino acid sequence identified as 1B and depicted in FIG.1A-1C. For example, an E1 polypeptide of genotype 1C can comprise anamino acid sequence having having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 192-383 of anamino acid sequence identified as 1C and depicted in FIG. 1A-1C.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence of an E1polypeptide depicted in FIG. 2A-2C. For example, an E1 polypeptide ofgenotype 2A can comprise an amino acid sequence having having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 192-383 of an amino acid sequence identified as 2A anddepicted in FIG. 2A-2C. For example, an E1 polypeptide of genotype 2Bcan comprise an amino acid sequence having having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids192-383 of an amino acid sequence identified as 2B and depicted in FIG.2A-2C.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the consensus E1 polypeptide aminoacid sequence depicted in FIG. 3A-3C.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence of an E1polypeptide depicted in FIG. 4A-4B. For example, an E1 polypeptide ofgenotype 7A can comprise an amino acid sequence having having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 192-383 of the amino acid sequence depicted in FIG. 4A-4B.

Variant E2

As noted above, a variant E2 polypeptide of an HCV E1/E2 heterodimer ofthe present disclosure comprises: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T cell epitopes (e.g.,one or more T-cell epitopes not present in an HCV E1 or an HCV E2polypeptide; e.g., one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 or an HCV E2 polypeptide). In somecases, the variant HCV E2 polypeptide comprises, in order from aminoterminus (N-terminus) to carboxyl terminus (C-terminus): i) an HCV E2polypeptide; and ii) a heterologous polypeptide comprising one or more Tcell epitopes. In some cases, the variant HCV E2 polypeptide comprises,in order from N-terminus to C-terminus: i) a heterologous polypeptidecomprising one or more T cell epitopes; and ii) an HCV E2 polypeptide.

In some cases, a variant E2 polypeptide of an HCV E1/E2 heterodimer ofthe present disclosure comprises from 1 to 10 amino acids at theN-terminus of the variant E2 polypeptide, which 1 to 10 amino acids arepart of a cleavable linker that remains following cleavage of apolyprotein precursor, as described below. For example, where thecleavable linker comprises the amino acid sequence LEVLFQGP (SEQ IDNO:5), the variant E2 polypeptide can comprise Gly-Pro residues at theN-terminus of the polypeptide, e.g., as depicted in FIG. 5A.

E2

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can have a length of fromabout 200 amino acids (aa) to about 250 aa, from about 250 aa to about275 aa, from about 275 aa to about 300 aa, from about 300 aa to about325 aa, from about 325 aa to about 350 aa, or from about 350 aa to about365 aa. In some cases, an E2 polypeptide suitable for inclusion in avariant E2 polypeptide of an HCV E1/E2 heterodimer of the presentdisclosure is a full-length HCV E1 polypeptide. In some cases, an E2polypeptide suitable for inclusion in a variant E1 polypeptide of an HCVE1/E2 heterodimer of the present disclosure is an HCV E2 ectodomainpolypeptide.

In FIG. 1A-AC, the amino acid sequence of E2 is amino acid 384 to aminoacid 746. In FIG. 2A-2B, the amino acid sequence of E2 is amino acid 384to amino acid 751. In FIG. 3A-3C, the amino acid sequence of E2 is aminoacid 385 to amino acid 754. In FIG. 4A-4B, the amino acid sequence of E2is amino acid 384 to amino acid 750. As used herein, an “E2 polypeptide”includes a precursor E2 protein, including the signal sequence; includesa mature E2 polypeptide which lacks this sequence; and includes an E2polypeptide with a heterologous signal sequence. An E2 polypeptide caninclude a C-terminal membrane anchor sequence which occurs atapproximately amino acid positions 715-730 and may extend as far asapproximately amino acid residue 746 (see, Lin et al., J. Virol. (1994)68:5063-5073).

In some cases, a E2 polypeptide suitable for inclusion in a variant E2polypeptide of an E1/E2 heterodimer of the present disclosure lacks aportion of its C-terminal region, e.g., from about amino acid 715 to theC-terminus; from about amino acid 625 to the C-terminus; from aboutamino acid 661 to the C-terminus; from about amino acid 655 to theC-terminus; from about amino acid 500 to the C-terminus, where the aminoacid numbering is with reference to the numbering in FIG. 1A-1C. See,e.g., U.S. Pat. No. 6,521,423.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence of an E2polypeptide depicted in FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG.4A-4B.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence of an E2polypeptide depicted in FIG. 1A-1C. For example, an E2 polypeptide ofgenotype 1 can comprise an amino acid sequence having having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 384-746 of an amino acid sequence depicted in FIG. 1A-1C.For example, an E2 polypeptide of genotype 1A can comprise an amino acidsequence having having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to amino acids 384-746 of an amino acidsequence identified as 1A and depicted in FIG. 1A-1C. For example, an E2polypeptide of genotype 1B can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 384-746 of an amino acid sequenceidentified as 1B and depicted in FIG. 1A-1C. For example, an E2polypeptide of genotype 1C can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 384-746 of an amino acid sequenceidentified as 1C and depicted in FIG. 1A-1C.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence of an E2polypeptide depicted in FIG. 2A-2C. For example, an E2 polypeptide cancomprise an amino acid sequence having having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids384-746 of an amino acid sequence depicted in FIG. 2A-2C. For example,an E2 polypeptide of genotype 2A can comprise an amino acid sequencehaving having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 384-751 of the “consensus” aminoacid sequence depicted in FIG. 2A-2C. For example, an E2 polypeptide ofgenotype 2B can comprise an amino acid sequence having having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 384-751 of the “consensus” amino acid sequence depicted inFIG. 2A-2C.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence of an E2polypeptide depicted in FIG. 3A-3C. For example, an E2 polypeptide ofgenotype 3 can comprise an amino acid sequence having having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 385-754 of an amino acid sequence depicted in FIG. 3A-3C.For example, an E2 polypeptide of genotype 3A can comprise an amino acidsequence having having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to amino acids 385-754 of an amino acidsequence identified as 3A and depicted in FIG. 3A-3C. For example, an E2polypeptide of genotype 3B can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 385-754 of the amino acid sequenceidentified as 3B and depicted in FIG. 3A-3C. For example, an E2polypeptide of genotype 3K can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 385-754 of the amino acid sequenceidentified as 3K and depicted in FIG. 3A-3C.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the amino acid sequence of the E2polypeptide depicted in FIG. 4A-4B. For example, an E2 polypeptide ofgenotype 7A can comprise an amino acid sequence having having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 384-750 of the amino acid sequence depicted in FIG. 4A-4B.

Heterologous Polypeptide

As noted above, in some embodiments, an E1/E2 heterodimer of the presentdisclosure comprises: a) a variant E1 polypeptide comprising: i) an HCVE1 polypeptide; and 2) a heterologous polypeptide comprising one or moreT cell epitopes (e.g., one or more T-cell epitopes not present in an HCVE1 or an HCV E2 polypeptide; e.g., one or more T-cell epitopes presentin an HCV polypeptide other than an HCV E1 or an HCV E2 polypeptide);and b) a variant E2 polypeptide comprising: i) an HCV E2 polypeptide;and 2) a heterologous polypeptide comprising one or more T cell epitopes(e.g., one or more T-cell epitopes not present in an HCV E1 or an HCV E2polypeptide; e.g., one or more T-cell epitopes present in an HCVpolypeptide other than an HCV E1 or an HCV E2 polypeptide).

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, or more than 10 (e.g., from 10 to 15, from 15 to 20,from 20 to 25, or from 25 to 30, or more than 30), T cell epitopes.T-cell epitopes are epitopes that, when presented with a majorhistocompatibility complex (MHC) (e.g., a human leukocyte antigen (HLA))Class I or MHC Class II molecule, are recognized and bound by a T-cellreceptor (TCR) present on a T cell surface. T-cell epitopes includeepitopes recognized by cytotoxic T cells (e.g., CD8⁺ T cells), andepitopes recognized by helper T cells (e.g., CD4⁺ T cells).

The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) anHCV NS4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5Apolypeptide; f) an HCV NS5B polypeptide; g) an HCV core polypeptide; orh) an HCV p7 polypeptide. In some cases, the one or more T-cell epitopesare T-cell epitopes present in an HCV NS3 polypeptide. In some cases,the heterologous polypeptide further comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) ameningococcal outer membrane protein. Other examples of strong T helperepitopes are diphtheria toxoid, tetanus toxoid, meningococcal outermembrane protein, or mutant diphtheria protein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127).

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS3 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS3 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS3 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS3 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS3 CD4⁺ T cell epitope and at least one HCV-NS3 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS3 CD4⁺ T-cell epitopes and 2 or more HCV-NS3 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS3 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS3 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS2 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS2 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS2 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS2 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS2 CD4⁺ T cell epitope and at least one HCV-NS2 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS2 CD4⁺ T-cell epitopes and 2 or more HCV-NS2 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS2 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS2 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS4A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS4A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS4A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS4A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS4A CD4⁺ T cell epitope and at least one HCV-NS4A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS4A CD4⁺ T-cell epitopes and 2 or more HCV-NS4A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS4A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS4A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5A CD4⁺ T cell epitope and at least one HCV-NS5A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5A CD4⁺ T-cell epitopes and 2 or more HCV-NS5A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5B T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5B T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5B T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5B T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5B CD4⁺ T cell epitope and at least one HCV-NS5B CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5B CD4⁺ T-cell epitopes and 2 or more HCV-NS5B CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5B CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5B CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-core T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-core T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-core T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-core CD4⁺ T cell epitope and at least one HCV-core CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-core CD4⁺ T-cell epitopes and 2 or more HCV-core CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-core CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-core CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-p7 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-p7 T-cell epitopes.In some cases, the heterologous polypeptide comprises 4 or more HCV-p7T-cell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD4⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core CD4⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises one or more HCV CD8⁺ Tcell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD8⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 CD8⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope. In some cases,the heterologous polypeptide comprises at least one HCV-p7 CD4⁺ T cellepitope and at least one HCV-p7 CD8⁺ T cell epitope. In some cases,heterologous polypeptide comprises 2 or more HCV-p7 CD4⁺ T-cell epitopesand 2 or more HCV-p7 CD8⁺ T-cell epitopes. In some cases, theheterologous polypeptide comprises 2, 3, 4, or 5 HCV-p7 CD4⁺ T-cellepitopes and 2, 3, 4, or 5 HCV-p7 CD8⁺ T-cell epitopes.

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, or 63, of the T-cell epitopes set out in FIG. 13A-13B. In somecases, the heterologous polypeptide comprises from 1 to 3, from 3 to 5,from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, or from 25 to30 of the T-cell epitopes set out in FIG. 13A-13B. For example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS3-3, NS3-4, and NS3-11 in FIG. 13A-13B and FIG. 15A-15N. Asanother example, in some cases, the heterologous polypeptide comprisesthe T-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 inFIG. 13A-13B and FIG. 15A-15N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-3,NS3-4, NS3-5, and NS3-11 in FIG. 13A-13B and FIG. 15A-15N. As anotherexample, in some cases, the heterologous polypeptide comprises theT-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated Core-1, Core-2, Core-3, Core-4, Core-5, Core-6,Core-7, Core-8, Core-9, Core-10, Core-11, Core-12, Core-13, Core-14,Core-16, Core-17, Core-18, Core-19, Core-20, Core-21, and Core-22 inFIG. 13A-13B and FIG. 15A-15N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-1,NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6, NS2-7, NS2-8,NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1,NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, andNS4b-10 in FIG. 13A-13B and FIG. 15A-15N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8,NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2,NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1,NS5a-2, NS5b-1, and NS5b-2 in FIG. 13A-13B and FIG. 15A-15N. In somecases, the T-cell epitopes are contiguous. In some cases, any two T-cellepitopes are separated by linkers (e.g., a linker having a length offrom 1 amino acid to about 50 amino acids, e.g., from 1 amino acid to 5amino acids (aa), from 5 aa to 10 aa, from 10 aa to 15 aa, from 15 aa to20 aa, from 20 aa to 25 aa, from 25 aa to 30 aa, from 30 aa to 40 aa, orfrom 40 aa to 50 aa).

In some cases, the heterologous polypeptide comprises at least one HCVCD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope, whereepitopes are conserved among HCV genotypes 1 and 2. In some cases, theheterologous polypeptide comprises at least one HCV CD4⁺ T cell epitopeand at least one HCV CD8⁺ T cell epitope, where epitopes are conservedamong HCV genotypes 1 and 3. In some cases, the heterologous polypeptidecomprises at least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺T cell epitope, where epitopes are conserved among HCV genotypes 1, 2,and 3. In some cases, the heterologous polypeptide comprises at leastone HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope,where epitopes are conserved among HCV genotypes 1, 2, 3, and 7. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope, where epitopesare conserved among HCV genotypes 1-7.

The heterologous polypeptide can have a length of from about 10 aminoacids to about 2000 amino acids; e.g., the heterologous polypeptide canhave a length of from 10 amino acids (aa) to 15 aa, from 15 aa to 20 aa,from 20 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to 75 aa, from 75aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aato 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to400 aa, from 450 aa to 500 aa, from 500 aa to 550 aa, from 550 aa to 600aa, from 600 aa to 650 aa, from 650 aa to 700 aa, from 700 aa to 750 aa,or from 750 aa to 800 aa. The heterologous polypeptide can have a lengthof from about 25 amino acids to about 2000 amino acids, e.g., from about25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa,from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aa to 250 aa,from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to 400 aa,from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to 700 aa,from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to 1000 aa,from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aa to 1300aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500 aa to1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from 1800 aato 1900 aa, or from 1900 aa to 2000 aa. The heterologous polypeptide canhave a length of from about 25 amino acids to about 3000 amino acids,e.g., from about 25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aato 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to700 aa, from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to1000 aa, from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aato 1300 aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500aa to 1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from1800 aa to 1900 aa, from 1900 aa to 2000 aa, from 2000 aa to 2250 aa,from 2250 aa to 2500 aa, from 2500 aa to 2750 aa, or from 2750 aa to3000 aa.

The heterologous polypeptide can have a length of from about 25 aminoacids to about 800 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from about 25 aminoacids to about 400 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, or from 350 aa to 400 aa. The heterologouspolypeptide can have a length of 25 amino acids (aa), 26 aa, 27 aa, 28aa, 29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa, 36 aa, 37 aa, 38aa, 39 aa, 40 aa, 41 aa, 42 aa, 43 aa, 44 aa, 45 aa, 46 aa, 47 aa, 48aa, 49 aa, or 50 aa. The heterologous polypeptide can have a length offrom about 100 amino acids (aa) to 800 aa, e.g., from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from 25 aa to 30 aa.The heterologous polypeptide can have a length of from 30 aa to 40 aa.The heterologous polypeptide can have a length of from 40 aa to 50 aa.The heterologous polypeptide can have a length of from 50 aa to 60 aa(e.g., 50 aa, 51 aa, 52, aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa,59 aa, or 60 aa). The heterologous polypeptide can have a length of from60 aa to 70 aa. The heterologous polypeptide can have a length of from65 aa to 75 aa (e.g., 65, 66, 67, 68, 69, 70, 71, 72, 7, 74, or 75 aa).The heterologous polypeptide can have a length of 70 aa. Theheterologous polypeptide can have a length of from 70 aa to 80 aa. Theheterologous polypeptide can have a length of from 80 aa to 90 aa. Theheterologous polypeptide can have a length of from 90 aa to 100 aa. Theheterologous polypeptide can have a length of from 100 aa to 105 aa(e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 aa). Theheterologous polypeptide can have a length of 100 aa. The heterologouspolypeptide can have a length of from 10 amino acids (aa) to 50 aa;e.g., from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to 25 aa,from 25 aa to 30 aa, from 30 aa to 35 aa, from 35 aa to 40 aa, from 40aa to 45 aa, or from 45 aa to 50 aa. The heterologous polypeptide canhave a length of from 10 amino acids (aa) to 20 aa, e.g., 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 aa.

HCV NS3 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS3 polypeptide. Examplesof T-cell epitopes present in NS3 polypeptides are depicted in FIG.15A-15N, FIG. 13B, and FIG. 14A-14B.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1). In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1); and has a length of from 25aa to 35 aa (e.g., 25 aa, 26 aa, 27 aa, 28 aa, 29 aa, 30 aa, 31 aa, 32aa, 33 aa, 34 aa, or 35 aa). In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1); and has alength of 29 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-3, NS3-4, and NS3-11 in FIG. 13B and FIG.15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2); andhas a length of from 45 amino acids to 60 amino acids (e.g., 45 aa, 46aa, 47 aa, 48 aa, 49 aa, 50 aa, 51 aa, 52 aa, 53 aa, 54 aa, 55 aa, 56aa, 57 aa, 58 aa, 59 aa, or 60 aa). In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2); andhas a length of 52 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-3, NS3-4, NS3-5, and NS3-11 in FIG. 13B and FIG.15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:3); and has a length of from 65 amino acids to 80 amino acids(e.g., 65 aa, 66 aa, 67 aa, 68 aa, 69 aa, 70 aa, 71 aa, 72 aa, 73 aa, 74aa, 75 aa, 76 aa, 77 aa, 78 aa, 79 aa, or 80 aa). In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:3); and has a length of 70 amino acids. Such a polytope caninclude NS3 T-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6,NS3-7, NS3-11, NS3-12, and NS3-13 in FIG. 13B and FIG. 15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4); and has a lengthof from 95 amino acids (aa) to 105 aa (e.g., 95 aa, 96 aa, 97 aa, 98 aa,99 aa, 100 aa, 101 aa, 102 aa, 103 aa, 104 aa, or 105 aa). In somecases, the heterologous polypeptide comprises an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4); and has a lengthof 100 amino acids. Such a polytope can include NS3 T-cell epitopesdesignated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 13B and FIG. 15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:9); and has a length of from 190 aminoacids (aa) to 200 aa (e.g., 190 aa, 191 aa, 192 aa, 193 aa, 194 aa, 195aa, 196 aa, 197 aa, 198 aa, 199 aa, or 200 aa. In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:9); and has a length of 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:10); and hasa length of from 215 amino acids (aa) to 235 aa (e.g., 215 aa, 216 aa,217 11, 218 aa, 219 aa, 220 aa, 221 aa, 222 aa, 223 aa, 224 aa, 225 aa,226 aa, 227 aa, 228 aa, 229 aa, 230 aa, 231 aa, 232 aa, 233 aa, 234 aa,or 235 aa). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:10); and hasa length of 228 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 13B and FIG. 15A-15N.

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1265-1279 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1309-1323 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1401-1415 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1402-1412 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1429-1439 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1464 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1453-1467 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1577-1591 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1306-1314 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1387-1394 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 1amino acids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12aa, 13 aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1405-1413 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1458 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1457-1465 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1610-1618 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

HCV NS2 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS2 polypeptide. Examplesof T-cell epitopes present in NS2 polypeptides are depicted in FIG.15A-15N, and FIG. 13A.

For example, the heterologous polypeptide can comprise an NS2 T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids955-974 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 975-994 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 985-1004 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1015-1034 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1035-1054 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 924-933 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 961-970 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 989-997 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 50 aa (e.g., from 10 aa to 25 aa, or from 25 aa to 50 aa) ofamino acids 955-1004 of the amino acid sequence designated “Consensus”in FIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa, orfrom 25 aa to 50 aa. In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 955-1004of the amino acid sequence designated “Consensus” in FIG. 16A-16L, or acorresponding HCV NS2 amino acid sequence of any HCV genotype; and has alength of about 50 amino acids.

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 553 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from 300 aato 400 aa, from 400 aa to 500 aa, or from 500 aa to 553 aa) of aminoacids 917-1469 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 and NS3 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa,from 25 aa to 50 aa, from 50 aa to 100 aa, from 100 aa to 200 aa, from200 aa to 300 aa, from 300 aa to 400 aa, from 400 aa to 500 aa, or from500 aa to 553 aa. In some cases, the heterologous polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 917-1469 of the aminoacid sequence designated “Consensus” in FIG. 16A-16L, or a correspondingHCV NS2 and NS3 amino acid sequence of any HCV genotype; and has alength of about 553 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 0%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11); andhas a length of from 50 amino acids to 60 amino acids (e.g., 50 aa, 51aa, 52 aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60 aa).In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11); andhas a length of 50 amino acids. Such a polytope can include NS2 T-cellepitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 in FIG. 13Aand FIG. 15A-15N.

HCV NS4A T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS4A polypeptide. Examplesof T-cell epitopes present in NS4A polypeptides are depicted in FIG.15A-15N and FIG. 13B.

The heterologous polypeptide can comprise an NS4A T cell epitopecomprising an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids1683-1692 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS4A amino acid sequence of any HCVgenotype; and the NS4A T-cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

HCV NS4B T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS4B polypeptide. Examplesof T-cell epitopes present in NS4B polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NS4B T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids1790-1801 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 12 aminoacids (aa) to 20 amino acids (e.g., 12 aa, 13 aa, 14 aa, 15 aa, 16 aa,17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1792-1802 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 11 aminoacids (aa) to 20 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15 aa,16 aa, 17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1898-1905 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 8 aminoacids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1921-1935 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1922-1941 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1928-1947 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1868-1876 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1927-1942 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 16 aminoacids (aa) to 20 amino acids (e.g., 16 aa, 17 aa, 18 aa, 19 aa, or 20aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1932-1940 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1948-1962 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

HCV NSSA T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NSSA polypeptide. Examplesof T-cell epitopes present in NSSA polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NSSA T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2218-2232 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NSSA amino acid sequence of any HCVgenotype; and the NSSA T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NSSA Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2309-2317 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS5A amino acid sequence of any HCVgenotype; and the NS5A T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV NS5B T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS5B polypeptide. Examplesof T-cell epitopes present in NS5B polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NS5B T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2847-2851 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS5B amino acid sequence of any HCVgenotype; and the NS5B T-cell epitope can have a length of from 5 aminoacids (aa) to 10 amino acids (e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10aa).

As another example, the heterologous polypeptide can comprise an NS5B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2602-2610 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NSSB amino acid sequence of any HCVgenotype; and the NSSB T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV Core T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV core polypeptide. Examplesof T-cell epitopes present in HCV Core polypeptides are depicted in FIG.15A-15N and FIG. 13A.

As one example, the heterologous polypeptide can comprise an HCV core Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1-20 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 11-30 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 21-40 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 39-63 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 23amino acids (aa) to 28 amino acids (e.g., 23 aa, 24 aa, 25 aa, 26 aa, 27aa, or 28 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 47-70 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 24amino acids (aa) to 29 amino acids (e.g., 24 aa, 25 aa, 26 aa, 27 aa, 28aa, or 29 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 61-80 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 71-90 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 81-100 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 91-110 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 101-115 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 111-130 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 125-139 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-150 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 151-170 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 161-180 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 35-44 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 43-51 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 51-59 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 129-137 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-140 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 150-158 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 154-162 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 168-176 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 177-187 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 178-187 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 191 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, or from 150 aato 191 aa) of amino acids 1-191 of the amino acid sequence designated“Consensus” in FIG. 16A-16L, or a corresponding HCV core amino acidsequence of any HCV genotype; and has a length of from 10 amino acids(aa) to 25 aa, from 25 aa to 50 aa, from 50 aa to 100 aa, or from 100 aato 150 aa, or from 150 aa to 191 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to amino acids1-191 of the amino acid sequence designated “Consensus” in FIG. 16A-16L,or a corresponding HCV core amino acid sequence of any HCV genotype; andhas a length of about 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLE DGVNYATGNLPG (SEQID NO:63); and has a length of from 171 amino acids (aa) to 180 aa(e.g., 171 aa, 172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa,179 aa, or 180 aa. In some cases, the heterologous polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLE DGVNYATGNLPG (SEQID NO:63); and has a length of 171 amino acids. Such a polytope caninclude core T-cell epitopes designated Core-1, Core-2, Core-3, Core-4,Core-5, Core-6, Core-7, Core-8, Core-9, Core-10, Core-11, Core-12,Core-13, Core-14, Core-16, Core-17, Core-18, Core-19, Core-20, Core-21,Core-22 in FIG. 13A and FIG. 15A-15N.

HCV p7 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV p7 polypeptide. Examplesof T-cell epitopes present in HCV p7 polypeptides are depicted in FIG.15A-15N or FIG. 13A.

As another example, the heterologous polypeptide can comprise an HCV p7T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 803-811 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV p7 amino acid sequence of any HCVgenotype; and the HCV p7 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

Polytopes Including HCV T-Cell Epitopes from More than One HCVPolypeptide Other than E1 and E2

As noted above, a heterologous polypeptide can include T-cell epitopesfrom more than one HCV polypeptide other than E1 and E2.

As one example, a heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTG DFDSVIDCN (SEQ IDNO:12); and has a length of from 550 amino acids (aa) to 560 aa (e.g.,550 aa, 551 aa, 552 aa, 553 aa, 554 aa, 555 aa, 556 aa, 557 aa, 558 aa,559 aa, or 560 aa). In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTG DFDSVIDCN (SEQ IDNO:12); and has a length of 553 amino acids. Such a polytope can includeT-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6,NS2-7, NS2-8, NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9,NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N.This polytope is also referred to as “TP553” (FIG. 14A-14D). In order toprevent self cleavage of the TP553 polytope (amino acids 917-1469) (FIG.15E-G) at the NS2-NS3 junction that is mediated by the catalytic domainof the NS2 protease (amino acids 917-1040), the histidine at position966 (H966), a critical residue for NS2 protease activity, is mutated toalanine (H966A) (FIG. 15E).

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aato 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa) the followingamino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to a contiguous stretch of from 25amino acids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa,from 200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa,from 350 aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa)of the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64); and has a length of from 25 amino acids (aa) to 50 aa,from 50 aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from300 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600aa to 700 aa, or from 700 aa to 778 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64); and has a length of 778 amino acids. Such a polytope caninclude T-cell epitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5,NS3-6, NS3-7, NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS2-14,NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8,NS4b-9, and NS4b-10 in FIG. 13B and FIG. 15A-15N.

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 1985 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 500 aa, from 500 aa to 750 aa, from 750aa to 1000 aa, from 1000 aa to 1500 aa, or from 1500 aa to 1985 aa) ofthe following amino acid sequence:

(SEQ ID NO: 13) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTV YHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVT RHADVIPVRRRGDSRGSLLSPRPISYLKGS A GGPLLCPAGHAVGIFRAAVCTRGV AKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAA YAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYG KFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPP GSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLV ALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQT VDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSV LCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHID AHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPT PLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCL STGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQ KALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPG NPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGA AIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGAL VVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARV TAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKL MPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTC RNMWSGTFPINTAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGM TTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQL PCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCT ANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVP AEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPP RKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAE SYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAE EQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEV KAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVW KDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYD VVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTE SDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVL TTSCGNTLTCYIKARAACRAAGLQDCTMLVCG NN LVVICESAGVQEDAASLRA FTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLA RAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEI YGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHR ARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR. 

In some cases, the heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGMTTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPPRKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGNNLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR (SEQ ID NO:13); and has a length of1985 amino acids. Such a polytope can include T-cell epitopes designatedNS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8, NS3-9, NS3-10,NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4,NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1, NS5a-2, NS5b-1,NS5b-2 in FIG. 13A-13B and FIG. 15A-15N.

Additional T-Cell Epitopes

As discussed above, an E1/E2 a heterodimeric polypeptide of the presentdisclosure includes: a) an HCV E2 polypeptide; and b) a variant HCV E1polypeptide comprising: i) an HCV E1 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide). The one or more T-cell epitopescan include one or more T-cell epitopes present in: a) an HCV NS3polypeptide; b) an HCV NS2 polypeptide; c) an HCV NS4A polypeptide; d)an HCV NS4B polypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5Bpolypeptide; g) an HCV core polypeptide; or h) an HCV p7 polypeptide. Insome cases, the one or more T-cell epitopes are T-cell epitopes presentin an HCV NS3 polypeptide. In some cases, the heterologous polypeptidecomprises one or more T cell epitopes present in: a) cholera toxin;and/or b) tetanus toxin; and/or c) diphtheria toxin; and/or d) ameningococcal outer membrane protein.

Thus, in some cases, a variant HCV E1 polypeptide of an E1/E2heterodimer of the present disclosure includes: a) an HCV E1polypeptide; and b) a heterologous polypeptide that comprises one ormore T-cell epitopes, where the one or more T-cell epitopes are T-cellepitopes present in: i) one or more of an HCV NS3 polypeptide, an HCVNS2 polypeptide, an HCV NS4A polypeptide, an HCV NS4B polypeptide, anHCV NS5A polypeptide, an HCV NS5B polypeptide, an HCV core polypeptide,and an HCV p7 polypeptide; and ii) one or more of cholera toxin ortoxoid, tetanus toxin or toxoid, diphtheria toxin or toxoid, and ameningococcal outer membrane protein.

A T helper tetanus toxin epitope or other bacterial T-cell epitope couldbe fused (e.g., by recombinant expression) or chemically conjugated tothe HCV polytope/E2 fusion protein and/or to the HCV polytope E1 fusionprotein of an E/E2 heterodimer of the present disclosure to furtherenhance both T and B cell responses to both the HCV polytope and E1/E2moieties. Alternatively, the whole or part of the detoxified toxin(“toxoid”) could be fused (e.g., by recombinant expression) orchemically conjugated to the HCV polytope/E1E2 protein, wherein specificamino acids of the toxins are mutated to render the toxins inactive,thereby generating toxoids. Methods of generating toxoids are well knownin the art. Examples of bacterial epitopes include the use of diphtheriatoxoid, meningococcal outer membrane protein, or mutant diphtheriaprotein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127)

In some cases, a suitable tetanus toxoid polypeptide comprises the aminoacid sequence QYIKANSKFIGIFE (SEQ ID NO:14). In some cases, a suitabletetanus toxoid polypeptide comprises the amino acid sequenceQYIKANSKFIGITE (SEQ ID NO:65).

In some cases, a heterologous polypeptide can comprise cholera toxin (ortoxoid) epitope. In some cases, a suitable heterologous polypeptidecomprising a cholera toxoid epitope comprises a fragment of choleratoxin-B subunit (CT-B), e.g., a fragment of from 5 amino acids to 25amino acids, or from 25 amino acids to 50 amino acids, of the followingamino acid sequence: MIKLKFGVFF TVLLSSAYAH GTPQNITDLC AEYHNTQIHTLNDKIFSYTE SLAGKREMAI ITFKNGATFQ VEVPGSQHID SQKKAIERMK DTLRIAYLTEAKVEKLCVWN NKTPHAIAAI SMAN (SEQ ID NO:15). In some cases, a suitableheterologous polypeptide comprising a cholera toxoid epitope comprisesthe following amino acid sequence: SLAGKREMAIITFKNGATFQVEVPG (SEQ IDNO:29).

In some cases, a heterologous polypeptide can comprise a tetanus toxin(or toxoid) T-cell epitope. In some cases, a suitable heterologouspolypeptide comprising a tetanus toxin T-cell epitope comprises theamino acid sequence: ILMQYIKANSKFIGI (SEQ ID NO:16); and has a length offrom 15 amino acids to 20 amino acids. In some cases, a suitableheterologous polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: VNNESSE (SEQ ID NO:17). In somecases, a suitable heterologous polypeptide comprising a tetanus toxinT-cell epitope comprises the amino acid sequence: PGINGKAIHLVNNESSE (SEQID NO:18). In some cases, a suitable heterologous polypeptide comprisinga tetanus toxin T-cell epitope comprises the amino acid sequence: PNRDIL(SEQ ID NO:19). In some cases, a suitable heterologous polypeptidecomprising a tetanus toxin T-cell epitope comprises the amino acidsequence: FIGITEL (SEQ ID NO:20). In some cases, a suitable tetanustoxin T-cell epitope comprises the amino acid sequence: SYFPSV (SEQ IDNO:21). In some cases, a suitable heterologous polypeptide comprising atetanus toxin T-cell epitope comprises the amino acid sequence:NSVDDALINSTKIYSYFPSV (SEQ ID NO:22). In some cases, a suitableheterologous polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: IDKISDVSTIVPYIGPALNI (SEQ ID NO:23).

In some cases, a heterologous polypeptide can comprise a diphtheriatoxin T-cell epitope In some cases, a suitable heterologous polypeptidecomprising a diphtheria toxin T-cell epitope comprises the amino acidsequence: QSIALSSLMVAQAIP (SEQ ID NO:24); and has a length of from 15amino acids to 20 amino acids. In some cases, a suitable heterologouspolypeptide comprising a diphtheria toxin T-cell epitope comprises theamino acid sequence: PVFAGANYAAWAVNVAQVI (SEQ ID NO:25). In some cases,a suitable heterologous polypeptide comprising a diphtheria toxin T-cellepitope comprises the amino acid sequence: VHHNTEEIVAQSIALSSLMV (SEQ IDNO:26). In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QSIALSSLMVAQAIPLVGEL (SEQ ID NO:66). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: VDIGFAAYNFVESIINLFQV (SEQ ID NO:67).In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QGESGHDIKITAENTPLPIA (SEQ ID NO:68). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: GVLLPTIPGKLDVNKSKTHI (SEQ ID NO:69).In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence ofCRM197 (see, e.g., Giannini et al. (1984) Nucl. Acids. Res. 12:4063).The amino acid sequence of CRM197 is provided above.

In some cases, a heterologous polypeptide can comprise a tetanus toxinT-cell epitope and a diphtheria toxin T-cell epitope. In some of thesecases, the heterologous polypeptide can comprise the amino acidsequence: IMQYIKANSKFIGIQSIALSSLMVAQ (SEQ ID NO:28); and can have alength of from 26 amino acids to 30 amino acids.

Additional Polypeptides

In any of the above-described embodiments, one or both of thepolypeptide chains of the E1/E2 heterodimer can include one or moreadditional polypeptides. For example, in some cases, the variant E1polypeptide or the variant E2 polypeptide can include an Ig Fcpolypeptide at the C-terminus of the variant E1 polypeptide or thevariant E2 polypeptide. Ig Fc polypeptides are known in the art, and aredescribed elsewhere herein.

II. Variant E2 Polypeptides

The present disclosure provides a variant HCV E2 polypeptide thatcomprises: a) an HCV E2 polypeptide; and b) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide). The heterologous polypeptide is also referredto as a “polytope.” A variant E2 polypeptide of the present disclosureis useful for inclusion in an E1/E2 heterodimer of the presentdisclosure.

The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) anHCV NS4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NSSApolypeptide; f) an HCV NSSB polypeptide; g) an HCV core polypeptide; orh) an HCV p7 polypeptide. In some cases, the one or more T-cell epitopesare T-cell epitopes present in an HCV NS3 polypeptide. In some cases,the heterologous polypeptide further comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) ameningococcal outer membrane protein. In some cases, a variant HCV E2polypeptide of the present disclosure includes: a) an HCV E2polypeptide; and b) a heterologous polypeptide that comprises one ormore T-cell epitopes, where the one or more T-cell epitopes are T-cellepitopes present in: i) one or more of an HCV NS3 polypeptide, an HCVNS2 polypeptide, an HCV NS4A polypeptide, an HCV NS4B polypeptide, anHCV NSSA polypeptide, an HCV NSSB polypeptide, an HCV core polypeptide,and an HCV p7 polypeptide; and ii) one or more of cholera toxin ortoxoid, tetanus toxin or toxoid, diphtheria toxin or toxoid, and ameningococcal outer membrane protein.

In some cases, the variant HCV E2 polypeptide comprises, in order fromN-terminus to C-terminus: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide comprising one or more T cell epitopes. In somecases, the variant HCV E2 polypeptide comprises, in order fromN-terminus to C-terminus: i) a heterologous polypeptide comprising oneor more T cell epitopes; and ii) an HCV E2 polypeptide.

E2

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofthe present disclosure can have a length of from about 200 amino acids(aa) to about 250 aa, from about 250 aa to about 275 aa, from about 275aa to about 300 aa, from about 300 aa to about 325 aa, from about 325 aato about 350 aa, or from about 350 aa to about 365 aa. The E2polypeptide can be a full-length HCV E2 polypeptide. The E2 polypeptidecan be an HCV E2 ectodomain polypeptide.

In FIG. 1A-AC, the amino acid sequence of E2 is amino acid 384 to aminoacid 746. In FIG. 2A-2B, the amino acid sequence of E2 is amino acid 384to amino acid 751. In FIG. 3A-3C, the amino acid sequence of E2 is aminoacid 385 to amino acid 754. In FIG. 4A-4B, the amino acid sequence of E2is amino acid 384 to amino acid 750. As used herein, an “E2 polypeptide”includes a precursor E2 protein, including the signal sequence; includesa mature E2 polypeptide which lacks this sequence; and includes an E2polypeptide with a heterologous signal sequence. An E2 polypeptide caninclude a C-terminal membrane anchor sequence which occurs atapproximately amino acid positions 715-730 and may extend as far asapproximately amino acid residue 746 (see, Lin et al., J. Virol. (1994)68:5063-5073).

In some cases, a E2 polypeptide suitable for inclusion in a variant E2polypeptide of the present disclosure lacks a portion of its C-terminalregion, e.g., from about amino acid 715 to the C-terminus; from aboutamino acid 625 to the C-terminus; from about amino acid 661 to theC-terminus; from about amino acid 655 to the C-terminus; from aboutamino acid 500 to the C-terminus, where the amino acid numbering is withreference to the numbering in FIG. 1A-1C. See, e.g., U.S. Pat. No.6,521,423.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence of an E2polypeptide depicted in FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG.4A-4B.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofthe present disclosure can comprise an amino acid sequence having havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.1A-1C. For example, an E2 polypeptide of genotype 1 can comprise anamino acid sequence having having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 384-746 of anamino acid sequence depicted in FIG. 1A-1C. For example, an E2polypeptide of genotype 1A can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 384-746 of an amino acid sequenceidentified as 1A and depicted in FIG. 1A-1C. For example, an E2polypeptide of genotype 1B can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 384-746 of an amino acid sequenceidentified as 1B and depicted in FIG. 1A-1C. For example, an E2polypeptide of genotype 1C can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 384-746 of an amino acid sequenceidentified as 1C and depicted in FIG. 1A-1C.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofthe present disclosure can comprise an amino acid sequence having havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.2A-2C. For example, an E2 polypeptide can comprise an amino acidsequence having having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to amino acids 384-746 of an amino acidsequence depicted in FIG. 2A-2C. For example, an E2 polypeptide ofgenotype 2A can comprise an amino acid sequence having having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 384-751 of the “consensus” amino acid sequence depicted inFIG. 2A-2C. For example, an E2 polypeptide of genotype 2B can comprisean amino acid sequence having having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 384-751 of the“consensus” amino acid sequence depicted in FIG. 2A-2C.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofan E1/E2 heterodimer of the present disclosure can comprise an aminoacid sequence having having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence of an E2polypeptide depicted in FIG. 3A-3C. For example, an E2 polypeptide ofgenotype 3 can comprise an amino acid sequence having having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 385-754 of an amino acid sequence depicted in FIG. 3A-3C.For example, an E2 polypeptide of genotype 3A can comprise an amino acidsequence having having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to amino acids 385-754 of an amino acidsequence identified as 3A and depicted in FIG. 3A-3C. For example, an E2polypeptide of genotype 3B can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 385-754 of the amino acid sequenceidentified as 3B and depicted in FIG. 3A-3C. For example, an E2polypeptide of genotype 3K can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 385-754 of the amino acid sequenceidentified as 3K and depicted in FIG. 3A-3C.

An E2 polypeptide suitable for inclusion in a variant E2 polypeptide ofthe present disclosure can comprise an amino acid sequence having havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the amino acid sequence of the E2 polypeptide depicted inFIG. 4A-4B. For example, an E2 polypeptide of genotype 7A can comprisean amino acid sequence having having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 384-750 of theamino acid sequence depicted in FIG. 4A-4B.

Heterologous Polypeptide

The heterologous polypeptide present in an E2 variant polypeptide of thepresent disclosure includes one or more T-cell epitopes.

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, or more than 10 (e.g., from 10 to 15, from 15 to 20,from 20 to 25, or from 25 to 30, or more than 30), T cell epitopes.T-cell epitopes are epitopes that, when presented with a majorhistocompatibility complex (MHC) (e.g., a human leukocyte antigen (HLA))Class I or MHC Class II molecule, are recognized and bound by a T-cellreceptor (TCR) present on a T cell surface. T-cell epitopes includeepitopes recognized by cytotoxic T cells (e.g., CD8⁺ T cells), andepitopes recognized by helper T cells (e.g., CD4⁺ T cells).

The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) anHCV NS4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NSSApolypeptide; f) an HCV NSSB polypeptide; g) an HCV core polypeptide; orh) an HCV p7 polypeptide. In some cases, the one or more T-cell epitopesare T-cell epitopes present in an HCV NS3 polypeptide. In some cases,the heterologous polypeptide further comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) ameningococcal outer membrane protein. Other examples of strong T helperepitopes are diphtheria toxoid, tetanus toxoid, meningococcal outermembrane protein, or mutant diphtheria protein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127).

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS3 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS3 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS3 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS3 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS3 CD4⁺ T cell epitope and at least one HCV-NS3 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS3 CD4⁺ T-cell epitopes and 2 or more HCV-NS3 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS3 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS3 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS2 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS2 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS2 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS2 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS2 CD4⁺ T cell epitope and at least one HCV-NS2 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS2 CD4⁺ T-cell epitopes and 2 or more HCV-NS2 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS2 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS2 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS4A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS4A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS4A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS4A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS4A CD4⁺ T cell epitope and at least one HCV-NS4A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS4A CD4⁺ T-cell epitopes and 2 or more HCV-NS4A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS4A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS4A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5A CD4⁺ T cell epitope and at least one HCV-NS5A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5A CD4⁺ T-cell epitopes and 2 or more HCV-NS5A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5B T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5B T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5B T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5B T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5B CD4⁺ T cell epitope and at least one HCV-NS5B CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5B CD4⁺ T-cell epitopes and 2 or more HCV-NS5B CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5B CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5B CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-core T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-core T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-core T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-core CD4⁺ T cell epitope and at least one HCV-core CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-core CD4⁺ T-cell epitopes and 2 or more HCV-core CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-core CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-core CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-p7 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-p7 T-cell epitopes.In some cases, the heterologous polypeptide comprises 4 or more HCV-p7T-cell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD4⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core CD4⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises one or more HCV CD8⁺ Tcell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD8⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 CD8⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope. In some cases,the heterologous polypeptide comprises at least one HCV-p7 CD4⁺ T cellepitope and at least one HCV-p7 CD8⁺ T cell epitope. In some cases,heterologous polypeptide comprises 2 or more HCV-p7 CD4⁺ T-cell epitopesand 2 or more HCV-p7 CD8⁺ T-cell epitopes. In some cases, theheterologous polypeptide comprises 2, 3, 4, or 5 HCV-p7 CD4⁺ T-cellepitopes and 2, 3, 4, or 5 HCV-p7 CD8⁺ T-cell epitopes.

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, or 63, of the T-cell epitopes set out in FIG. 13A-13B. In somecases, the heterologous polypeptide comprises from 1 to 3, from 3 to 5,from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, or from 25 to30 of the T-cell epitopes set out in FIG. 13A-13B. For example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS3-3, NS3-4, and NS3-11 in FIG. 13A-13B and FIG. 15A-15N. Asanother example, in some cases, the heterologous polypeptide comprisesthe T-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 inFIG. 13A-13B and FIG. 15A-15N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-3,NS3-4, NS3-5, and NS3-11 in FIG. 13A-13B and FIG. 15A-15N. As anotherexample, in some cases, the heterologous polypeptide comprises theT-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated Core-1, Core-2, Core-3, Core-4, Core-5, Core-6,Core-7, Core-8, Core-9, Core-10, Core-11, Core-12, Core-13, Core-14,Core-16, Core-17, Core-18, Core-19, Core-20, Core-21, and Core-22 inFIG. 13A-13B and FIG. 15A-15N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-1,NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6, NS2-7, NS2-8,NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1,NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, andNS4b-10 in FIG. 13A-13B and FIG. 15A-15N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8,NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2,NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1,NS5a-2, NS5b-1, and NS5b-2 in FIG. 13A-13B and FIG. 15A-15N. In somecases, the T-cell epitopes are contiguous. In some cases, any two T-cellepitopes are separated by linkers (e.g., a linker having a length offrom 1 amino acid to about 50 amino acids, e.g., from 1 amino acid to 5amino acids (aa), from 5 aa to 10 aa, from 10 aa to 15 aa, from 15 aa to20 aa, from 20 aa to 25 aa, from 25 aa to 30 aa, from 30 aa to 40 aa, orfrom 40 aa to 50 aa).

In some cases, the heterologous polypeptide comprises at least one HCVCD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope, whereepitopes are conserved among HCV genotypes 1 and 2. In some cases, theheterologous polypeptide comprises at least one HCV CD4⁺ T cell epitopeand at least one HCV CD8⁺ T cell epitope, where epitopes are conservedamong HCV genotypes 1 and 3. In some cases, the heterologous polypeptidecomprises at least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺T cell epitope, where epitopes are conserved among HCV genotypes 1, 2,and 3. In some cases, the heterologous polypeptide comprises at leastone HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope,where epitopes are conserved among HCV genotypes 1, 2, 3, and 7. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope, where epitopesare conserved among HCV genotypes 1-7.

The heterologous polypeptide can have a length of from about 10 aminoacids to about 2000 amino acids; e.g., the heterologous polypeptide canhave a length of from 10 amino acids (aa) to 15 aa, from 15 aa to 20 aa,from 20 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to 75 aa, from 75aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aato 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to400 aa, from 450 aa to 500 aa, from 500 aa to 550 aa, from 550 aa to 600aa, from 600 aa to 650 aa, from 650 aa to 700 aa, from 700 aa to 750 aa,or from 750 aa to 800 aa. The heterologous polypeptide can have a lengthof from about 25 amino acids to about 2000 amino acids, e.g., from about25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa,from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aa to 250 aa,from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to 400 aa,from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to 700 aa,from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to 1000 aa,from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aa to 1300aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500 aa to1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from 1800 aato 1900 aa, or from 1900 aa to 2000 aa. The heterologous polypeptide canhave a length of from about 25 amino acids to about 3000 amino acids,e.g., from about 25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aato 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to700 aa, from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to1000 aa, from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aato 1300 aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500aa to 1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from1800 aa to 1900 aa, from 1900 aa to 2000 aa, from 2000 aa to 2250 aa,from 2250 aa to 2500 aa, from 2500 aa to 2750 aa, or from 2750 aa to3000 aa.

The heterologous polypeptide can have a length of from about 25 aminoacids to about 800 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from about 25 aminoacids to about 400 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, or from 350 aa to 400 aa. The heterologouspolypeptide can have a length of 25 amino acids (aa), 26 aa, 27 aa, 28aa, 29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa, 36 aa, 37 aa, 38aa, 39 aa, 40 aa, 41 aa, 42 aa, 43 aa, 44 aa, 45 aa, 46 aa, 47 aa, 48aa, 49 aa, or 50 aa. The heterologous polypeptide can have a length offrom about 100 amino acids (aa) to 800 aa, e.g., from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from 25 aa to 30 aa.The heterologous polypeptide can have a length of from 30 aa to 40 aa.The heterologous polypeptide can have a length of from 40 aa to 50 aa.The heterologous polypeptide can have a length of from 50 aa to 60 aa(e.g., 50 aa, 51 aa, 52, aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa,59 aa, or 60 aa). The heterologous polypeptide can have a length of from60 aa to 70 aa. The heterologous polypeptide can have a length of from65 aa to 75 aa (e.g., 65, 66, 67, 68, 69, 70, 71, 72, 7, 74, or 75 aa).The heterologous polypeptide can have a length of 70 aa. Theheterologous polypeptide can have a length of from 70 aa to 80 aa. Theheterologous polypeptide can have a length of from 80 aa to 90 aa. Theheterologous polypeptide can have a length of from 90 aa to 100 aa. Theheterologous polypeptide can have a length of from 100 aa to 105 aa(e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 aa). Theheterologous polypeptide can have a length of 100 aa. The heterologouspolypeptide can have a length of from 10 amino acids (aa) to 50 aa;e.g., from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to 25 aa,from 25 aa to 30 aa, from 30 aa to 35 aa, from 35 aa to 40 aa, from 40aa to 45 aa, or from 45 aa to 50 aa. The heterologous polypeptide canhave a length of from 10 amino acids (aa) to 20 aa, e.g., 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 aa.

HCV NS3 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS3 polypeptide. Examplesof T-cell epitopes present in NS3 polypeptides are depicted in FIG.15A-15N, FIG. 13B, and FIG. 14A-14B.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1). In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1); and has a length of from 25aa to 35 aa (e.g., 25 aa, 26 aa, 27 aa, 28 aa, 29 aa, 30 aa, 31 aa, 32aa, 33 aa, 34 aa, or 35 aa). In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1); and has alength of 29 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-3, NS3-4, and NS3-11 in FIG. 13B and FIG.15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2); andhas a length of from 45 amino acids to 60 amino acids (e.g., 45 aa, 46aa, 47 aa, 48 aa, 49 aa, 50 aa, 51 aa, 52 aa, 53 aa, 54 aa, 55 aa, 56aa, 57 aa, 58 aa, 59 aa, or 60 aa). In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2); andhas a length of 52 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-3, NS3-4, NS3-5, and NS3-11 in FIG. 13B and FIG.15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:3); and has a length of from 65 amino acids to 80 amino acids(e.g., 65 aa, 66 aa, 67 aa, 68 aa, 69 aa, 70 aa, 71 aa, 72 aa, 73 aa, 74aa, 75 aa, 76 aa, 77 aa, 78 aa, 79 aa, or 80 aa). In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:3); and has a length of 70 amino acids. Such a polytope caninclude NS3 T-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6,NS3-7, NS3-11, NS3-12, and NS3-13 in FIG. 13B and FIG. 15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4); and has a lengthof from 95 amino acids (aa) to 105 aa (e.g., 95 aa, 96 aa, 97 aa, 98 aa,99 aa, 100 aa, 101 aa, 102 aa, 103 aa, 104 aa, or 105 aa). In somecases, the heterologous polypeptide comprises an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4); and has a lengthof 100 amino acids. Such a polytope can include NS3 T-cell epitopesdesignated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 13B and FIG. 15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:9); and has a length of from 190 aminoacids (aa) to 200 aa (e.g., 190 aa, 191 aa, 192 aa, 193 aa, 194 aa, 195aa, 196 aa, 197 aa, 198 aa, 199 aa, or 200 aa. In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:9); and has a length of 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:10); and hasa length of from 215 amino acids (aa) to 235 aa (e.g., 215 aa, 216 aa,217 11, 218 aa, 219 aa, 220 aa, 221 aa, 222 aa, 223 aa, 224 aa, 225 aa,226 aa, 227 aa, 228 aa, 229 aa, 230 aa, 231 aa, 232 aa, 233 aa, 234 aa,or 235 aa). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:10); and hasa length of 228 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 13B and FIG. 15A-15N.

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1265-1279 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1309-1323 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1401-1415 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1402-1412 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1429-1439 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1464 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1453-1467 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1577-1591 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1306-1314 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1387-1394 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 1amino acids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12aa, 13 aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1405-1413 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1458 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1457-1465 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1610-1618 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

HCV NS2 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS2 polypeptide. Examplesof T-cell epitopes present in NS2 polypeptides are depicted in FIG.15A-15N, and FIG. 13A.

For example, the heterologous polypeptide can comprise an NS2 T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids955-974 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 975-994 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 985-1004 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1015-1034 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1035-1054 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 924-933 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 961-970 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 989-997 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 50 aa (e.g., from 10 aa to 25 aa, or from 25 aa to 50 aa) ofamino acids 955-1004 of the amino acid sequence designated “Consensus”in FIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa, orfrom 25 aa to 50 aa. In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 955-1004of the amino acid sequence designated “Consensus” in FIG. 16A-16L, or acorresponding HCV NS2 amino acid sequence of any HCV genotype; and has alength of about 50 amino acids.

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 553 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from 300 aato 400 aa, from 400 aa to 500 aa, or from 500 aa to 553 aa) of aminoacids 917-1469 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 and NS3 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa,from 25 aa to 50 aa, from 50 aa to 100 aa, from 100 aa to 200 aa, from200 aa to 300 aa, from 300 aa to 400 aa, from 400 aa to 500 aa, or from500 aa to 553 aa. In some cases, the heterologous polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 917-1469 of the aminoacid sequence designated “Consensus” in FIG. 16A-16L, or a correspondingHCV NS2 and NS3 amino acid sequence of any HCV genotype; and has alength of about 553 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 0%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11); andhas a length of from 50 amino acids to 60 amino acids (e.g., 50 aa, 51aa, 52 aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60 aa).In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11); andhas a length of 50 amino acids. Such a polytope can include NS2 T-cellepitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 in FIG. 13Aand FIG. 15A-15N.

HCV NS4A T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS4A polypeptide. Examplesof T-cell epitopes present in NS4A polypeptides are depicted in FIG.15A-15N and FIG. 13B.

The heterologous polypeptide can comprise an NS4A T cell epitopecomprising an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids1683-1692 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS4A amino acid sequence of any HCVgenotype; and the NS4A T-cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

HCV NS4B T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS4B polypeptide. Examplesof T-cell epitopes present in NS4B polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NS4B T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids1790-1801 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 12 aminoacids (aa) to 20 amino acids (e.g., 12 aa, 13 aa, 14 aa, 15 aa, 16 aa,17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1792-1802 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 11 aminoacids (aa) to 20 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15 aa,16 aa, 17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1898-1905 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 8 aminoacids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1921-1935 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1922-1941 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1928-1947 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1868-1876 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1927-1942 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 16 aminoacids (aa) to 20 amino acids (e.g., 16 aa, 17 aa, 18 aa, 19 aa, or 20aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1932-1940 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1948-1962 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

HCV NS5A T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS5A polypeptide. Examplesof T-cell epitopes present in NS5A polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NS5A T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2218-2232 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS5A amino acid sequence of any HCVgenotype; and the NS5A T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NS5A Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2309-2317 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NSSA amino acid sequence of any HCVgenotype; and the NSSA T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV NSSB T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NSSB polypeptide. Examplesof T-cell epitopes present in NSSB polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NSSB T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2847-2851 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS5B amino acid sequence of any HCVgenotype; and the NS5B T-cell epitope can have a length of from 5 aminoacids (aa) to 10 amino acids (e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10aa).

As another example, the heterologous polypeptide can comprise an NS5B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2602-2610 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS5B amino acid sequence of any HCVgenotype; and the NS5B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV Core T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV core polypeptide. Examplesof T-cell epitopes present in HCV Core polypeptides are depicted in FIG.15A-15N and FIG. 13A.

As one example, the heterologous polypeptide can comprise an HCV core Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1-20 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 11-30 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 21-40 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 39-63 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 23amino acids (aa) to 28 amino acids (e.g., 23 aa, 24 aa, 25 aa, 26 aa, 27aa, or 28 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 47-70 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 24amino acids (aa) to 29 amino acids (e.g., 24 aa, 25 aa, 26 aa, 27 aa, 28aa, or 29 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 61-80 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 71-90 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 81-100 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 91-110 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 101-115 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 111-130 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 125-139 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-150 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 151-170 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 161-180 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 35-44 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 43-51 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 51-59 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 129-137 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-140 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 150-158 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 154-162 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 168-176 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 177-187 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 178-187 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 191 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, or from 150 aato 191 aa) of amino acids 1-191 of the amino acid sequence designated“Consensus” in FIG. 16A-16L, or a corresponding HCV core amino acidsequence of any HCV genotype; and has a length of from 10 amino acids(aa) to 25 aa, from 25 aa to 50 aa, from 50 aa to 100 aa, or from 100 aato 150 aa, or from 150 aa to 191 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to amino acids1-191 of the amino acid sequence designated “Consensus” in FIG. 16A-16L,or a corresponding HCV core amino acid sequence of any HCV genotype; andhas a length of about 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLE DGVNYATGNLPG (SEQID NO:63); and has a length of from 171 amino acids (aa) to 180 aa(e.g., 171 aa, 172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa,179 aa, or 180 aa. In some cases, the heterologous polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLE DGVNYATGNLPG (SEQID NO:63); and has a length of 171 amino acids. Such a polytope caninclude core T-cell epitopes designated Core-1, Core-2, Core-3, Core-4,Core-5, Core-6, Core-7, Core-8, Core-9, Core-10, Core-11, Core-12,Core-13, Core-14, Core-16, Core-17, Core-18, Core-19, Core-20, Core-21,Core-22 in FIG. 13A and FIG. 15A-15N.

HCV p7 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV p7 polypeptide. Examplesof T-cell epitopes present in HCV p7 polypeptides are depicted in FIG.15A-15N or FIG. 13A.

As another example, the heterologous polypeptide can comprise an HCV p7T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 803-811 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV p7 amino acid sequence of any HCVgenotype; and the HCV p7 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

Polytopes Including HCV T-Cell Epitopes from More than One HCVPolypeptide Other than E1 and E2

As noted above, a heterologous polypeptide can include T-cell epitopesfrom more than one HCV polypeptide other than E1 and E2.

As one example, a heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTG DFDSVIDCN (SEQ IDNO:12); and has a length of from 550 amino acids (aa) to 560 aa (e.g.,550 aa, 551 aa, 552 aa, 553 aa, 554 aa, 555 aa, 556 aa, 557 aa, 558 aa,559 aa, or 560 aa). In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTG DFDSVIDCN (SEQ IDNO:12); and has a length of 553 amino acids. Such a polytope can includeT-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6,NS2-7, NS2-8, NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9,NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N.This polytope is also referred to as “TP553” (FIG. 14A-14D). In order toprevent self cleavage of the TP553 polytope (amino acids 917-1469) (FIG.15E-G) at the NS2-NS3 junction that is mediated by the catalytic domainof the NS2 protease (amino acids 917-1040), the histidine at position966 (H966), a critical residue for NS2 protease activity, is mutated toalanine (H966A) (FIG. 15E).

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aato 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa) the followingamino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to a contiguous stretch of from 25amino acids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa,from 200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa,from 350 aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa)of the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64); and has a length of from 25 amino acids (aa) to 50 aa,from 50 aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from300 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600aa to 700 aa, or from 700 aa to 778 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64); and has a length of 778 amino acids. Such a polytope caninclude T-cell epitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5,NS3-6, NS3-7, NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS2-14,NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8,NS4b-9, and NS4b-10 in FIG. 13B and FIG. 15A-15N.

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 1985 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 500 aa, from 500 aa to 750 aa, from 750aa to 1000 aa, from 1000 aa to 1500 aa, or from 1500 aa to 1985 aa) ofthe following amino acid sequence:

(SEQ ID NO: 13) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTV YHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVT RHADVIPVRRRGDSRGSLLSPRPISYLKGS A GGPLLCPAGHAVGIFRAAVCTRGV AKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAA YAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYG KFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPP GSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLV ALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQT VDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSV LCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHID AHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPT PLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCL STGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQ KALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPG NPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGA AIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGAL VVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARV TAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKL MPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTC RNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGM TTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQL PCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCT ANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVP AEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPP RKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAE SYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAE EQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEV KAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVW KDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYD VVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTE SDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVL TTSCGNTLTCYIKARAACRAAGLQDCTMLVCG NN LVVICESAGVQEDAASLRA FTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLA RAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEI YGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHR ARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR. 

In some cases, the heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGMTTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPPRKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGNNLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR (SEQ ID NO:13); and has a length of1985 amino acids. Such a polytope can include T-cell epitopes designatedNS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8, NS3-9, NS3-10,NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4,NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1, NS5a-2, NS5b-1,NS5b-2 in FIG. 13A-13B and FIG. 15A-15N.

Additional T-Cell Epitopes

As discussed above, an E1/E2 a heterodimeric polypeptide of the presentdisclosure includes: a) an HCV E1 polypeptide; and b) a variant HCV E2polypeptide comprising: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide). The one or more T-cell epitopescan include one or more T-cell epitopes present in: a) an HCV NS3polypeptide; b) an HCV NS2 polypeptide; c) an HCV NS4A polypeptide; d)an HCV NS4B polypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5Bpolypeptide; g) an HCV core polypeptide; or h) an HCV p7 polypeptide. Insome cases, the one or more T-cell epitopes are T-cell epitopes presentin an HCV NS3 polypeptide. In some cases, the heterologous polypeptidecomprises one or more T cell epitopes present in: a) cholera toxin;and/or b) tetanus toxin; and/or c) diphtheria toxin.

Thus, in some cases, a variant HCV polypeptide of an E1/E2 heterodimerof the present disclosure includes: a) an HCV E2 polypeptide; and b) aheterologous polypeptide that comprises one or more T-cell epitopes,where the one or more T-cell epitopes are T-cell epitopes present in: i)one or more of an HCV NS3 polypeptide, an HCV NS2 polypeptide, an HCVNS4A polypeptide, an HCV NS4B polypeptide, an HCV NS5A polypeptide, anHCV NS5B polypeptide, an HCV core polypeptide, and an HCV p7polypeptide; and ii) one or more of cholera toxin or toxoid, tetanustoxin or toxoid, diphtheria toxin or toxoid, and a meningococcal outermembrane protein.

In some cases, a heterologous polypeptide can comprise a tetanus toxinT-cell epitope. In some cases, a suitable heterologous polypeptidecomprising a tetanus toxin T-cell epitope comprises the amino acidsequence: ILMQYIKANSKFIGI (SEQ ID NO:16); and has a length of from 15amino acids to 20 amino acids. In some cases, a suitable heterologouspolypeptide comprising a tetanus toxin T-cell epitope comprises theamino acid sequence: VNNESSE (SEQ ID NO:17). In some cases, a suitableheterologous polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: PGINGKAIHLVNNESSE (SEQ ID NO:18). Insome cases, a suitable heterologous polypeptide comprising a tetanustoxin T-cell epitope comprises the amino acid sequence: PNRDIL (SEQ IDNO:19). In some cases, a suitable heterologous polypeptide comprising atetanus toxin T-cell epitope comprises the amino acid sequence: FIGITEL(SEQ ID NO:20). In some cases, a suitable tetanus toxin T-cell epitopecomprises the amino acid sequence: SYFPSV (SEQ ID NO:21). In some cases,a suitable heterologous polypeptide comprising a tetanus toxin T-cellepitope comprises the amino acid sequence: NSVDDALINSTKIYSYFPSV (SEQ IDNO:22). In some cases, a suitable heterologous polypeptide comprising atetanus toxin T-cell epitope comprises the amino acid sequence:IDKISDVSTIVPYIGPALNI (SEQ ID NO:23).

In some cases, a heterologous polypeptide can comprise a diphtheriatoxin T-cell epitope In some cases, a suitable heterologous polypeptidecomprising a diphtheria toxin T-cell epitope comprises the amino acidsequence: QSIALSSLMVAQAIP (SEQ ID NO:24); and has a length of from 15amino acids to 20 amino acids. In some cases, a suitable heterologouspolypeptide comprising a diphtheria toxin T-cell epitope comprises theamino acid sequence: PVFAGANYAAWAVNVAQVI (SEQ ID NO:25). In some cases,a suitable heterologous polypeptide comprising a diphtheria toxin T-cellepitope comprises the amino acid sequence: VHHNTEEIVAQSIALSSLMV (SEQ IDNO:26). In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QSIALSSLMVAQAIPLVGEL (SEQ ID NO:66). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: VDIGFAAYNFVESIINLFQV (SEQ ID NO:67).In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QGESGHDIKITAENTPLPIA (SEQ ID NO:68). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: GVLLPTIPGKLDVNKSKTHI (SEQ ID NO:69).

In some cases, a heterologous polypeptide can comprise a tetanus toxinT-cell epitope and a diphtheria toxin T-cell epitope. In some of thesecases, the heterologous polypeptide can comprise the amino acidsequence: IMQYIKANSKFIGIQSIALSSLMVAQ (SEQ ID NO:28); and can have alength of from 26 amino acids to 30 amino acids.

Additional Polypeptides

In any of the above-described embodiments, the variant E2 polypeptidecan include one or more additional polypeptides. For example, in somecases, the variant E2 polypeptide includes an Ig Fc polypeptide at theC-terminus of variant E2 polypeptide. As another example, in some cases,the variant E2 polypeptide includes an Ig Fc polypeptide at theN-terminus of variant E2 polypeptide. Ig Fc polypeptides are known inthe art, and are described elsewhere herein.

III. Variant E1 Polypeptides

The present disclosure provides a variant HCV E1 polypeptide thatcomprises: a) an HCV E1 polypeptide; and b) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide). The heterologous polypeptide is also referredto as a “polytope.” A variant E1 polypeptide of the present disclosureis useful for including in an E1/E2 heterodimer of the presentdisclosure.

The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) anHCV NS4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5Apolypeptide; f) an HCV NS5B polypeptide; g) an HCV core polypeptide; orh) an HCV p7 polypeptide. In some cases, the one or more T-cell epitopesare T-cell epitopes present in an HCV NS3 polypeptide. In some cases,the heterologous polypeptide further comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) ameningococcal outer membrane protein. In some cases, a variant HCV E1polypeptide of the present disclosure includes: a) an HCV E1polypeptide; and b) a heterologous polypeptide that comprises one ormore T-cell epitopes, where the one or more T-cell epitopes are T-cellepitopes present in: i) one or more of an HCV NS3 polypeptide, an HCVNS2 polypeptide, an HCV NS4A polypeptide, an HCV NS4B polypeptide, anHCV NS5A polypeptide, an HCV NS5B polypeptide, an HCV core polypeptide,and an HCV p7 polypeptide; and ii) one or more of cholera toxin ortoxoid, tetanus toxin or toxoid, diphtheria toxin or toxoid, and ameningococcal outer membrane protein.

In some cases, the variant HCV E1 polypeptide comprises, in order fromN-terminus to C-terminus: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide comprising one or more T cell epitopes. In somecases, the variant HCV E1 polypeptide comprises, in order fromN-terminus to C-terminus: i) a heterologous polypeptide comprising oneor more T cell epitopes; and ii) an HCV E1 polypeptide.

E1

An HCV E1 polypeptide suitable for inclusion in a variant E1 polypeptideof the present disclosure can have a length of from about 150 aminoacids (aa) to about 175 aa, from about 175 aa to about 195 aa, fromabout 131 aa to about 175 aa, or from about 175 aa to about 193 aa. TheE1 polypeptide can be a full-length HCV E1 polypeptide. The E1polypeptide can be an HCV E1 ectodomain polypeptide.

In FIG. 1A-1C, the amino acid sequence of E1 is amino acid 192 to aminoacid 383. In FIG. 2A-2C, the amino acid sequence of E1 is amino acid 192to amino acid 383. In FIG. 3A-3C, the amino acid sequence of E1 is aminoacid 192 to amino acid 384. In FIG. 4A-4B, the amino acid sequence of E1is amino acid 192 to amino acid 383. Amino acids at around 170 throughapproximately 191 serve as a signal sequence for E1. As used herein, “E1polypeptide” includes a precursor E1 protein, including the signalsequence; includes a mature E1 polypeptide which lacks this sequence;and includes an E1 polypeptide with a heterologous signal sequence. AnE1 polypeptide can include a C-terminal membrane anchor sequence whichoccurs at approximately amino acid positions 360-383 (see, e.g., WO96/04301). In some cases, a suitable E1 polypeptide lacks a C-terminalportion that includes a transmembrane region. For example, in somecases, a suitable E1 polypeptide lacks the C-terminal portion from aminoacid 330 to amino acid 384, or from amino acid 360 to amino acid 384. E1polypeptides can be an E1 polypeptide of any genotype, subtype orisolate of HCV. E1 polypeptides of genotype 1 and E1 polypeptides ofgenotype 3 are included in an E1/E2 heterodimer of the presentdisclosure.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofthe present disclosure can comprise an amino acid sequence having havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E1 polypeptide depicted in FIG.1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG. 4A-4B.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofthe present disclosure can comprise an amino acid sequence having havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E1 polypeptide depicted in FIG.1A-1C. For example, an E1 polypeptide of genotype 1A can comprise anamino acid sequence having having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 192-383 of anamino acid sequence identified as 1A and depicted in FIG. 1A-1C. Forexample, an E1 polypeptide of genotype 1B can comprise an amino acidsequence having having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to amino acids 192-383 of an amino acidsequence identified as 1B and depicted in FIG. 1A-1C. For example, an E1polypeptide of genotype 1C can comprise an amino acid sequence havinghaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 192-383 of an amino acid sequenceidentified as 1C and depicted in FIG. 1A-1C.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofthe present disclosure can comprise an amino acid sequence having havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E1 polypeptide depicted in FIG.2A-2C. For example, an E1 polypeptide of genotype 2A can comprise anamino acid sequence having having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 192-383 of anamino acid sequence identified as 2A and depicted in FIG. 2A-2C. Forexample, an E1 polypeptide of genotype 2B can comprise an amino acidsequence having having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to amino acids 192-383 of an amino acidsequence identified as 2B and depicted in FIG. 2A-2C.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofthe present disclosure can comprise an amino acid sequence having havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the consensus E1 polypeptide amino acid sequence depicted inFIG. 3A-3C.

An E1 polypeptide suitable for inclusion in a variant E1 polypeptide ofthe present disclosure can comprise an amino acid sequence having havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E1 polypeptide depicted in FIG.4A-4B. For example, an E1 polypeptide of genotype 7A can comprise anamino acid sequence having having at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 192-383 of theamino acid sequence depicted in FIG. 4A-4B.

Heterologous Polypeptide

The heterologous polypeptide present in an E1 variant polypeptide of thepresent disclosure includes one or more T-cell epitopes.

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, or more than 10 (e.g., from 10 to 15, from 15 to 20,from 20 to 25, or from 25 to 30, or more than 30), T cell epitopes.T-cell epitopes are epitopes that, when presented with a majorhistocompatibility complex (MHC) (e.g., a human leukocyte antigen (HLA))Class I or MHC Class II molecule, are recognized and bound by a T-cellreceptor (TCR) present on a T cell surface. T-cell epitopes includeepitopes recognized by cytotoxic T cells (e.g., CD8⁺ T cells), andepitopes recognized by helper T cells (e.g., CD4⁺ T cells).

The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) anHCV NS4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5Apolypeptide; f) an HCV NS5B polypeptide; g) an HCV core polypeptide; orh) an HCV p7 polypeptide. In some cases, the one or more T-cell epitopesare T-cell epitopes present in an HCV NS3 polypeptide. In some cases,the heterologous polypeptide further comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) ameningococcal outer membrane protein. Other examples of strong T helperepitopes are diphtheria toxoid, tetanus toxoid, meningococcal outermembrane protein, or mutant diphtheria protein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127).

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS3 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS3 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS3 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS3 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS3 CD4⁺ T cell epitope and at least one HCV-NS3 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS3 CD4⁺ T-cell epitopes and 2 or more HCV-NS3 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS3 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS3 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS2 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS2 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS2 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS2 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS2 CD4⁺ T cell epitope and at least one HCV-NS2 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS2 CD4⁺ T-cell epitopes and 2 or more HCV-NS2 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS2 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS2 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS4A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS4A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS4A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS4A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS4A CD4⁺ T cell epitope and at least one HCV-NS4A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS4A CD4⁺ T-cell epitopes and 2 or more HCV-NS4A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS4A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS4A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5A CD4⁺ T cell epitope and at least one HCV-NS5A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5A CD4⁺ T-cell epitopes and 2 or more HCV-NS5A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5B T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5B T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5B T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5B T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5B CD4⁺ T cell epitope and at least one HCV-NS5B CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5B CD4⁺ T-cell epitopes and 2 or more HCV-NS5B CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5B CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5B CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-core T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-core T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-core T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-core CD4⁺ T cell epitope and at least one HCV-core CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-core CD4⁺ T-cell epitopes and 2 or more HCV-core CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-core CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-core CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-p7 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-p7 T-cell epitopes.In some cases, the heterologous polypeptide comprises 4 or more HCV-p7T-cell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD4⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core CD4⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises one or more HCV CD8⁺ Tcell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD8⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 CD8⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope. In some cases,the heterologous polypeptide comprises at least one HCV-p7 CD4⁺ T cellepitope and at least one HCV-p7 CD8⁺ T cell epitope. In some cases,heterologous polypeptide comprises 2 or more HCV-p7 CD4⁺ T-cell epitopesand 2 or more HCV-p7 CD8⁺ T-cell epitopes. In some cases, theheterologous polypeptide comprises 2, 3, 4, or 5 HCV-p7 CD4⁺ T-cellepitopes and 2, 3, 4, or 5 HCV-p7 CD8⁺ T-cell epitopes.

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, or 63, of the T-cell epitopes set out in FIG. 13A-13B. In somecases, the heterologous polypeptide comprises from 1 to 3, from 3 to 5,from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, or from 25 to30 of the T-cell epitopes set out in FIG. 13A-13B. For example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS3-3, NS3-4, and NS3-11 in FIG. 13A-13B and FIG. 15A-15N. Asanother example, in some cases, the heterologous polypeptide comprisesthe T-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 inFIG. 13A-13B and FIG. 15A-15N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-3,NS3-4, NS3-5, and NS3-11 in FIG. 13A-13B and FIG. 15A-15N. As anotherexample, in some cases, the heterologous polypeptide comprises theT-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated Core-1, Core-2, Core-3, Core-4, Core-5, Core-6,Core-7, Core-8, Core-9, Core-10, Core-11, Core-12, Core-13, Core-14,Core-16, Core-17, Core-18, Core-19, Core-20, Core-21, and Core-22 inFIG. 13A-13B and FIG. 15A-15N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-1,NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6, NS2-7, NS2-8,NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1,NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, andNS4b-10 in FIG. 13A-13B and FIG. 15A-15N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8,NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2,NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1,NS5a-2, NS5b-1, and NS5b-2 in FIG. 13A-13B and FIG. 15A-15N. In somecases, the T-cell epitopes are contiguous. In some cases, any two T-cellepitopes are separated by linkers (e.g., a linker having a length offrom 1 amino acid to about 50 amino acids, e.g., from 1 amino acid to 5amino acids (aa), from 5 aa to 10 aa, from 10 aa to 15 aa, from 15 aa to20 aa, from 20 aa to 25 aa, from 25 aa to 30 aa, from 30 aa to 40 aa, orfrom 40 aa to 50 aa).

In some cases, the heterologous polypeptide comprises at least one HCVCD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope, whereepitopes are conserved among HCV genotypes 1 and 2. In some cases, theheterologous polypeptide comprises at least one HCV CD4⁺ T cell epitopeand at least one HCV CD8⁺ T cell epitope, where epitopes are conservedamong HCV genotypes 1 and 3. In some cases, the heterologous polypeptidecomprises at least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺T cell epitope, where epitopes are conserved among HCV genotypes 1, 2,and 3. In some cases, the heterologous polypeptide comprises at leastone HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope,where epitopes are conserved among HCV genotypes 1, 2, 3, and 7. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope, where epitopesare conserved among HCV genotypes 1-7.

The heterologous polypeptide can have a length of from about 10 aminoacids to about 2000 amino acids; e.g., the heterologous polypeptide canhave a length of from 10 amino acids (aa) to 15 aa, from 15 aa to 20 aa,from 20 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to 75 aa, from 75aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aato 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to400 aa, from 450 aa to 500 aa, from 500 aa to 550 aa, from 550 aa to 600aa, from 600 aa to 650 aa, from 650 aa to 700 aa, from 700 aa to 750 aa,or from 750 aa to 800 aa. The heterologous polypeptide can have a lengthof from about 25 amino acids to about 2000 amino acids, e.g., from about25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa,from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aa to 250 aa,from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to 400 aa,from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to 700 aa,from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to 1000 aa,from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aa to 1300aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500 aa to1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from 1800 aato 1900 aa, or from 1900 aa to 2000 aa. The heterologous polypeptide canhave a length of from about 25 amino acids to about 3000 amino acids,e.g., from about 25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aato 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to700 aa, from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to1000 aa, from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aato 1300 aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500aa to 1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from1800 aa to 1900 aa, from 1900 aa to 2000 aa, from 2000 aa to 2250 aa,from 2250 aa to 2500 aa, from 2500 aa to 2750 aa, or from 2750 aa to3000 aa.

The heterologous polypeptide can have a length of from about 25 aminoacids to about 800 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from about 25 aminoacids to about 400 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, or from 350 aa to 400 aa. The heterologouspolypeptide can have a length of 25 amino acids (aa), 26 aa, 27 aa, 28aa, 29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa, 36 aa, 37 aa, 38aa, 39 aa, 40 aa, 41 aa, 42 aa, 43 aa, 44 aa, 45 aa, 46 aa, 47 aa, 48aa, 49 aa, or 50 aa. The heterologous polypeptide can have a length offrom about 100 amino acids (aa) to 800 aa, e.g., from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from 25 aa to 30 aa.The heterologous polypeptide can have a length of from 30 aa to 40 aa.The heterologous polypeptide can have a length of from 40 aa to 50 aa.The heterologous polypeptide can have a length of from 50 aa to 60 aa(e.g., 50 aa, 51 aa, 52, aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa,59 aa, or 60 aa). The heterologous polypeptide can have a length of from60 aa to 70 aa. The heterologous polypeptide can have a length of from65 aa to 75 aa (e.g., 65, 66, 67, 68, 69, 70, 71, 72, 7, 74, or 75 aa).The heterologous polypeptide can have a length of 70 aa. Theheterologous polypeptide can have a length of from 70 aa to 80 aa. Theheterologous polypeptide can have a length of from 80 aa to 90 aa. Theheterologous polypeptide can have a length of from 90 aa to 100 aa. Theheterologous polypeptide can have a length of from 100 aa to 105 aa(e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 aa). Theheterologous polypeptide can have a length of 100 aa. The heterologouspolypeptide can have a length of from 10 amino acids (aa) to 50 aa;e.g., from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to 25 aa,from 25 aa to 30 aa, from 30 aa to 35 aa, from 35 aa to 40 aa, from 40aa to 45 aa, or from 45 aa to 50 aa. The heterologous polypeptide canhave a length of from 10 amino acids (aa) to 20 aa, e.g., 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 aa.

HCV NS3 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS3 polypeptide. Examplesof T-cell epitopes present in NS3 polypeptides are depicted in FIG.15A-15N, FIG. 13B, and FIG. 14A-14B.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1). In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1); and has a length of from 25aa to 35 aa (e.g., 25 aa, 26 aa, 27 aa, 28 aa, 29 aa, 30 aa, 31 aa, 32aa, 33 aa, 34 aa, or 35 aa). In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1); and has alength of 29 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-3, NS3-4, and NS3-11 in FIG. 13B and FIG.15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2); andhas a length of from 45 amino acids to 60 amino acids (e.g., 45 aa, 46aa, 47 aa, 48 aa, 49 aa, 50 aa, 51 aa, 52 aa, 53 aa, 54 aa, 55 aa, 56aa, 57 aa, 58 aa, 59 aa, or 60 aa). In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2); andhas a length of 52 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-3, NS3-4, NS3-5, and NS3-11 in FIG. 13B and FIG.15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:3); and has a length of from 65 amino acids to 80 amino acids(e.g., 65 aa, 66 aa, 67 aa, 68 aa, 69 aa, 70 aa, 71 aa, 72 aa, 73 aa, 74aa, 75 aa, 76 aa, 77 aa, 78 aa, 79 aa, or 80 aa). In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:3); and has a length of 70 amino acids. Such a polytope caninclude NS3 T-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6,NS3-7, NS3-11, NS3-12, and NS3-13 in FIG. 13B and FIG. 15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4); and has a lengthof from 95 amino acids (aa) to 105 aa (e.g., 95 aa, 96 aa, 97 aa, 98 aa,99 aa, 100 aa, 101 aa, 102 aa, 103 aa, 104 aa, or 105 aa). In somecases, the heterologous polypeptide comprises an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4); and has a lengthof 100 amino acids. Such a polytope can include NS3 T-cell epitopesdesignated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 13B and FIG. 15A-15N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:9); and has a length of from 190 aminoacids (aa) to 200 aa (e.g., 190 aa, 191 aa, 192 aa, 193 aa, 194 aa, 195aa, 196 aa, 197 aa, 198 aa, 199 aa, or 200 aa. In some cases, theheterologous polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:9); and has a length of 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:10); and hasa length of from 215 amino acids (aa) to 235 aa (e.g., 215 aa, 216 aa,217 11, 218 aa, 219 aa, 220 aa, 221 aa, 222 aa, 223 aa, 224 aa, 225 aa,226 aa, 227 aa, 228 aa, 229 aa, 230 aa, 231 aa, 232 aa, 233 aa, 234 aa,or 235 aa). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:10); and hasa length of 228 amino acids. Such a polytope can include NS3 T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 13B and FIG. 15A-15N.

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1265-1279 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1309-1323 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1401-1415 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1402-1412 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1429-1439 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1464 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1453-1467 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1577-1591 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1306-1314 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1387-1394 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 1amino acids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12aa, 13 aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1405-1413 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1458 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1457-1465 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1610-1618 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

HCV NS2 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS2 polypeptide. Examplesof T-cell epitopes present in NS2 polypeptides are depicted in FIG.15A-15N, and FIG. 13A.

For example, the heterologous polypeptide can comprise an NS2 T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids955-974 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 975-994 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 985-1004 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1015-1034 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1035-1054 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 924-933 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 961-970 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 989-997 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 50 aa (e.g., from 10 aa to 25 aa, or from 25 aa to 50 aa) ofamino acids 955-1004 of the amino acid sequence designated “Consensus”in FIG. 16A-16L, or a corresponding HCV NS2 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa, orfrom 25 aa to 50 aa. In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 955-1004of the amino acid sequence designated “Consensus” in FIG. 16A-16L, or acorresponding HCV NS2 amino acid sequence of any HCV genotype; and has alength of about 50 amino acids.

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 553 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from 300 aato 400 aa, from 400 aa to 500 aa, or from 500 aa to 553 aa) of aminoacids 917-1469 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS2 and NS3 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa,from 25 aa to 50 aa, from 50 aa to 100 aa, from 100 aa to 200 aa, from200 aa to 300 aa, from 300 aa to 400 aa, from 400 aa to 500 aa, or from500 aa to 553 aa. In some cases, the heterologous polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 917-1469 of the aminoacid sequence designated “Consensus” in FIG. 16A-16L, or a correspondingHCV NS2 and NS3 amino acid sequence of any HCV genotype; and has alength of about 553 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 0%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11). Insome cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11); andhas a length of from 50 amino acids to 60 amino acids (e.g., 50 aa, 51aa, 52 aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60 aa).In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:11); andhas a length of 50 amino acids. Such a polytope can include NS2 T-cellepitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 in FIG. 13Aand FIG. 15A-15N.

HCV NS4A T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS4A polypeptide. Examplesof T-cell epitopes present in NS4A polypeptides are depicted in FIG.15A-15N and FIG. 13B.

The heterologous polypeptide can comprise an NS4A T cell epitopecomprising an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids1683-1692 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS4A amino acid sequence of any HCVgenotype; and the NS4A T-cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

HCV NS4B T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS4B polypeptide. Examplesof T-cell epitopes present in NS4B polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NS4B T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids1790-1801 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 12 aminoacids (aa) to 20 amino acids (e.g., 12 aa, 13 aa, 14 aa, 15 aa, 16 aa,17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1792-1802 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 11 aminoacids (aa) to 20 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15 aa,16 aa, 17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1898-1905 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 8 aminoacids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1921-1935 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1922-1941 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1928-1947 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1868-1876 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1927-1942 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 16 aminoacids (aa) to 20 amino acids (e.g., 16 aa, 17 aa, 18 aa, 19 aa, or 20aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1932-1940 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1948-1962 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

HCV NSSA T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NSSA polypeptide. Examplesof T-cell epitopes present in NSSA polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NSSA T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2218-2232 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NSSA amino acid sequence of any HCVgenotype; and the NSSA T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NSSA Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2309-2317 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NS5A amino acid sequence of any HCVgenotype; and the NS5A T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV NS5B T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV NS5B polypeptide. Examplesof T-cell epitopes present in NS5B polypeptides are depicted in FIG.15A-15N and FIG. 13B.

As one example, the heterologous polypeptide can comprise an NS5B T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2847-2851 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV NS5B amino acid sequence of any HCVgenotype; and the NS5B T-cell epitope can have a length of from 5 aminoacids (aa) to 10 amino acids (e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10aa).

As another example, the heterologous polypeptide can comprise an NS5B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2602-2610 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV NSSB amino acid sequence of any HCVgenotype; and the NSSB T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV Core T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV core polypeptide. Examplesof T-cell epitopes present in HCV Core polypeptides are depicted in FIG.15A-15N and FIG. 13A.

As one example, the heterologous polypeptide can comprise an HCV core Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1-20 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 11-30 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 21-40 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 39-63 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 23amino acids (aa) to 28 amino acids (e.g., 23 aa, 24 aa, 25 aa, 26 aa, 27aa, or 28 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 47-70 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 24amino acids (aa) to 29 amino acids (e.g., 24 aa, 25 aa, 26 aa, 27 aa, 28aa, or 29 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 61-80 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 71-90 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 81-100 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 91-110 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 101-115 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 111-130 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 125-139 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-150 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 151-170 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 161-180 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 35-44 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 43-51 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 51-59 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 129-137 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-140 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 150-158 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 154-162 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 168-176 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 177-187 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 178-187 of the amino acid sequence designated “Consensus” inFIG. 16A-16L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 191 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, or from 150 aato 191 aa) of amino acids 1-191 of the amino acid sequence designated“Consensus” in FIG. 16A-16L, or a corresponding HCV core amino acidsequence of any HCV genotype; and has a length of from 10 amino acids(aa) to 25 aa, from 25 aa to 50 aa, from 50 aa to 100 aa, or from 100 aato 150 aa, or from 150 aa to 191 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to amino acids1-191 of the amino acid sequence designated “Consensus” in FIG. 16A-16L,or a corresponding HCV core amino acid sequence of any HCV genotype; andhas a length of about 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLE DGVNYATGNLPG (SEQID NO:63); and has a length of from 171 amino acids (aa) to 180 aa(e.g., 171 aa, 172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa,179 aa, or 180 aa. In some cases, the heterologous polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLE DGVNYATGNLPG (SEQID NO:63); and has a length of 171 amino acids. Such a polytope caninclude core T-cell epitopes designated Core-1, Core-2, Core-3, Core-4,Core-5, Core-6, Core-7, Core-8, Core-9, Core-10, Core-11, Core-12,Core-13, Core-14, Core-16, Core-17, Core-18, Core-19, Core-20, Core-21,Core-22 in FIG. 13A and FIG. 15A-15N.

HCV p7 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an E2 variantpolypeptide of an E1/E2 heterodimer of the present disclosure includesone or more T-cell epitopes present in an HCV p7 polypeptide. Examplesof T-cell epitopes present in HCV p7 polypeptides are depicted in FIG.15A-15N or FIG. 13A.

As another example, the heterologous polypeptide can comprise an HCV p7T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 803-811 of the amino acid sequence designated “Consensus” in FIG.16A-16L, or a corresponding HCV p7 amino acid sequence of any HCVgenotype; and the HCV p7 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

Polytopes Including HCV T-Cell Epitopes from More than One HCVPolypeptide Other than E1 and E2

As noted above, a heterologous polypeptide can include T-cell epitopesfrom more than one HCV polypeptide other than E1 and E2.

As one example, a heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTG DFDSVIDCN (SEQ IDNO:12); and has a length of from 550 amino acids (aa) to 560 aa (e.g.,550 aa, 551 aa, 552 aa, 553 aa, 554 aa, 555 aa, 556 aa, 557 aa, 558 aa,559 aa, or 560 aa). In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTG DFDSVIDCN (SEQ IDNO:12); and has a length of 553 amino acids. Such a polytope can includeT-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6,NS2-7, NS2-8, NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9,NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 13A-13B and FIG. 15A-15N.This polytope is also referred to as “TP553” (FIG. 14A-14D). In order toprevent self cleavage of the TP553 polytope (amino acids 917-1469) (FIG.15E-G) at the NS2-NS3 junction that is mediated by the catalytic domainof the NS2 protease (amino acids 917-1040), the histidine at position966 (H966), a critical residue for NS2 protease activity, is mutated toalanine (H966A) (FIG. 15E).

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aato 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa) the followingamino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64). In some cases, the heterologous polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to a contiguous stretch of from 25amino acids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa,from 200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa,from 350 aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa)of the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64); and has a length of from 25 amino acids (aa) to 50 aa,from 50 aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from300 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600aa to 700 aa, or from 700 aa to 778 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:64); and has a length of 778 amino acids. Such a polytope caninclude T-cell epitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5,NS3-6, NS3-7, NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS2-14,NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8,NS4b-9, and NS4b-10 in FIG. 13B and FIG. 15A-15N.

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 1985 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 500 aa, from 500 aa to 750 aa, from 750aa to 1000 aa, from 1000 aa to 1500 aa, or from 1500 aa to 1985 aa) ofthe following amino acid sequence:

(SEQ ID NO: 13) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTV YHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVT RHADVIPVRRRGDSRGSLLSPRPISYLKGS A GGPLLCPAGHAVGIFRAAVCTRGV AKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAA YAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYG KFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPP GSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLV ALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQT VDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSV LCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHID AHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPT PLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCL STGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQ KALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPG NPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGA AIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGAL VVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARV TAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKL MPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTC RNMWSGTFPINTAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGM TTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQL PCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCT ANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVP AEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPP RKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAE SYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAE EQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEV KAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVW KDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYD VVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTE SDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVL TTSCGNTLTCYIKARAACRAAGLQDCTMLVCG NN LVVICESAGVQEDAASLRA FTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLA RAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEI YGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHR ARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR. 

In some cases, the heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGMTTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPPRKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGNNLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR (SEQ ID NO:13); and has a length of1985 amino acids. Such a polytope can include T-cell epitopes designatedNS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8, NS3-9, NS3-10,NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4,NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1, NS5a-2, NS5b-1,NS5b-2 in FIG. 13A-13B and FIG. 15A-15N.

Additional T-Cell Epitopes

As discussed above, the one or more T-cell epitopes present in aheterologous polypeptide can include one or more T-cell epitopes presentin: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) an HCV NS4Apolypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5A polypeptide; f)an HCV NS5B polypeptide; g) an HCV core polypeptide; or h) an HCV p7polypeptide. In some cases, the one or more T-cell epitopes are T-cellepitopes present in an HCV NS3 polypeptide. In some cases, theheterologous polypeptide comprises one or more T cell epitopes presentin: a) cholera toxin; and/or b) tetanus toxin; and/or c) diphtheriatoxin; and/or d) a meningococcal outer membrane protein.

Thus, in some cases, a variant HCV polypeptide of includes: a) an HCV E1polypeptide; and b) a heterologous polypeptide that comprises one ormore T-cell epitopes, where the one or more T-cell epitopes are T-cellepitopes present in: i) one or more of an HCV NS3 polypeptide, an HCVNS2 polypeptide, an HCV NS4A polypeptide, an HCV NS4B polypeptide, anHCV NS5A polypeptide, an HCV NS5B polypeptide, an HCV core polypeptide,and an HCV p7 polypeptide; and ii) one or more of cholera toxin ortoxoid, tetanus toxin or toxoid, diphtheria toxin or toxoid, and ameningococcal outer membrane protein.

A T helper tetanus toxin epitope or other bacterial T-cell epitope couldbe fused (e.g., by recombinant expression) or chemically conjugated tothe HCV polytope/E2 fusion protein and/or to the HCV polytope E1 fusionprotein of an E1/E2 heterodimer of the present disclosure to furtherenhance both T and B cell responses to both the HCV polytope and E1/E2moieties. Alternatively, the whole or part of the detoxified toxin(“toxoid”) could be fused (e.g., by recombinant expression) orchemically conjugated to the HCV polytope/E1E2 protein, wherein specificamino acids of the toxins are mutated to render the toxins inactive,thereby generating toxoids. Methods of generating toxoids are well knownin the art. Examples of bacterial epitopes include the use of diphtheriatoxoid, meningococcal outer membrane protein, or mutant diphtheriaprotein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127)

In some cases, a suitable tetanus toxoid polypeptide comprises the aminoacid sequence QYIKANSKFIGIFE (SEQ ID NO:14). In some cases, a suitabletetanus toxoid polypeptide comprises the amino acid sequenceQYIKANSKFIGITE (SEQ ID NO:65).

In some cases, a heterologous polypeptide can comprise cholera toxin (ortoxoid) epitope. In some cases, a suitable heterologous polypeptidecomprising a cholera toxoid epitope comprises a fragment of choleratoxin-B subunit (CT-B), e.g., a fragment of from 5 amino acids to 25amino acids, or from 25 amino acids to 50 amino acids, of the followingamino acid sequence: MIKLKFGVFF TVLLSSAYAH GTPQNITDLC AEYHNTQIHTLNDKIFSYTE SLAGKREMAI ITFKNGATFQ VEVPGSQHID SQKKAIERMK DTLRIAYLTEAKVEKLCVWN NKTPHAIAAI SMAN (SEQ ID NO:15). In some cases, a suitableheterologous polypeptide comprising a cholera toxoid epitope comprisesthe following amino acid sequence: SLAGKREMAIITFKNGATFQVEVPG (SEQ IDNO:29).

In some cases, a heterologous polypeptide can comprise a tetanus toxin(or toxoid) T-cell epitope. In some cases, a suitable heterologouspolypeptide comprising a tetanus toxin T-cell epitope comprises theamino acid sequence: ILMQYIKANSKFIGI (SEQ ID NO:16); and has a length offrom 15 amino acids to 20 amino acids. In some cases, a suitableheterologous polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: VNNESSE (SEQ ID NO:17). In somecases, a suitable heterologous polypeptide comprising a tetanus toxinT-cell epitope comprises the amino acid sequence: PGINGKAIHLVNNESSE (SEQID NO:18). In some cases, a suitable heterologous polypeptide comprisinga tetanus toxin T-cell epitope comprises the amino acid sequence: PNRDIL(SEQ ID NO:19). In some cases, a suitable heterologous polypeptidecomprising a tetanus toxin T-cell epitope comprises the amino acidsequence: FIGITEL (SEQ ID NO:20). In some cases, a suitable tetanustoxin T-cell epitope comprises the amino acid sequence: SYFPSV (SEQ IDNO:21). In some cases, a suitable heterologous polypeptide comprising atetanus toxin T-cell epitope comprises the amino acid sequence:NSVDDALINSTKIYSYFPSV (SEQ ID NO:22). In some cases, a suitableheterologous polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: IDKISDVSTIVPYIGPALNI (SEQ ID NO:23).

In some cases, a heterologous polypeptide can comprise a diphtheriatoxin T-cell epitope In some cases, a suitable heterologous polypeptidecomprising a diphtheria toxin T-cell epitope comprises the amino acidsequence: QSIALSSLMVAQAIP (SEQ ID NO:24); and has a length of from 15amino acids to 20 amino acids. In some cases, a suitable heterologouspolypeptide comprising a diphtheria toxin T-cell epitope comprises theamino acid sequence: PVFAGANYAAWAVNVAQVI (SEQ ID NO:25). In some cases,a suitable heterologous polypeptide comprising a diphtheria toxin T-cellepitope comprises the amino acid sequence: VHHNTEEIVAQSIALSSLMV (SEQ IDNO:26). In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QSIALSSLMVAQAIPLVGEL (SEQ ID NO:66). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: VDIGFAAYNFVESIINLFQV (SEQ ID NO:67).In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QGESGHDIKITAENTPLPIA (SEQ ID NO:68). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: GVLLPTIPGKLDVNKSKTHI (SEQ ID NO:69).In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence ofCRM197 (see, e.g., Giannini et al. (1984) Nucl. Acids. Res. 12:4063).The amino acid sequence of CRM197 is provided above.

In some cases, a heterologous polypeptide can comprise a tetanus toxinT-cell epitope and a diphtheria toxin T-cell epitope. In some of thesecases, the heterologous polypeptide can comprise the amino acidsequence: IMQYIKANSKFIGIQSIALSSLMVAQ (SEQ ID NO:28);

and can have a length of from 26 amino acids to 30 amino acids.

Additional Polypeptides

In any of the above-described embodiments, the variant E1 polypeptidecan include one or more additional polypeptides. For example, in somecases, the variant E1 polypeptide includes an Ig Fc polypeptide at theC-terminus of variant E1 polypeptide. As another example, the variant E1polypeptide includes an Ig Fc polypeptide at the N-terminus of variantE1 polypeptide. Ig Fc polypeptides are known in the art, and aredescribed elsewhere herein.

IV. Nucleic Acids Encoding a Variant HCV E1 Polypeptide, a Variant HCVE2 Polypeptide, or an E/E2 Heterodimer

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a variant HCV E2 polypeptide of the presentdisclosure. The present disclosure provides a nucleic acid comprising anucleotide sequence encoding a variant HCV E1 polypeptide of the presentdisclosure. The present disclosure provides a nucleic acid comprising anucleotide sequence an E1/E2 heterodimer of the present disclosure.

As described below, a variant HCV E1 polypeptide, a variant HCV E2polypeptide, or a heterodimer comprising: i) a variant HCV E2polypeptide and an HCV E1 polypeptide; ii) a variant HCV E1 polypeptideand an HCV E2 polypeptide; or iii) a variant HCV E1 polypeptide and avariant HCV E2 polypeptide, can be encoded by a nucleotide sequence of anucleic acid of the present disclosure. Where an Ig Fc region is at theC-terminus of the encoded heterodimer, a proteolytically cleavablelinker can be positioned between the Fc region and the polypeptide atthe N-terminus of the Ig Fc region.

IV(A). Variant E2 and E1/E2 Heterodimers Comprising a Variant E2Polypeptide

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a variant HCV E2 polypeptide of the presentdisclosure, where the variant HCV E2 polypeptide comprises: i) an HCV E2polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). The present disclosure provides a nucleic acid comprisinga nucleotide sequence encoding a polyprotein comprising a variant E2polypeptide of the present disclosure. In some cases, the nucleic acidis present in an expression vector. Thus, the present disclosureprovides a recombinant expression vector comprising a nucleotidesequence encoding a variant E2 polypeptide of the present disclosure,where the variant HCV E2 polypeptide comprises: i) an HCV E2polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide).

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding a variant E2 polypeptide of the presentdisclosure, where the variant HCV E2 polypeptide comprises: i) an HCV E2polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the nucleotide sequence encoding thevariant E2 polypeptide is operably linked to a transcription controlelement, e.g., a promoter that is functional in a eukaryotic cell.Suitable promoters include, e.g., a cytomegalovirus (CMV) promoter, anSV40 promoter, and the like. In some cases, the nucleic acid is presentin an expression vector (e.g., a lentivirus vector; an adenoassociatedvirus vector; an adenovirus vector; a retroviral vector; a non-viralvector; etc.). In some cases, the nucleic acid is present in anexpression vector, where the expression vector is a non-viral vector.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding a fusion protein comprising, in order fromN-terminus to C-terminus: a) an immunoglobulin (Ig) Fc region; and b) avariant E2 polypeptide of the present disclosure, where the variant HCVE2 polypeptide comprises: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E2 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In some cases,the variant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E2 polypeptide. In some cases, thenucleotide sequence encoding the fusion protein is operably linked to atranscription control element, e.g., a promoter that is functional in aeukaryotic cell. Suitable promoters include, e.g., a CMV promoter, anSV40 promoter, and the like. In some cases, the nucleic acid is presentin an expression vector (e.g., a lentivirus vector; an adenoassociatedvirus vector; an adenovirus vector; a retroviral vector; a non-viralvector; etc.). In some cases, a nucleic acid of the present disclosurecomprises a nucleotide sequence encoding a fusion protein comprising, inorder from N-terminus to C-terminus: a) an Ig Fc region; b) aproteolytically cleavable linker; and c) a variant E2 polypeptide of thepresent disclosure, where the variant HCV E2 polypeptide comprises: i)an HCV E2 polypeptide; and ii) a heterologous polypeptide that comprisesone or more T-cell epitopes (e.g., one or more T cell epitopes presentin an HCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E2 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E2 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E2 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E2polypeptide. In some cases, the nucleotide sequence encoding the fusionprotein is operably linked to a transcription control element, e.g., apromoter that is functional in a eukaryotic cell. Suitable promotersinclude, e.g., a CMV promoter, an SV40 promoter, and the like. In somecases, the nucleic acid is present in an expression vector (e.g., alentivirus vector; an adenoassociated virus vector; an adenovirusvector; a retroviral vector; a non-viral vector; etc.).

The proteolytically cleavable linker can include a protease recognitionsequence recognized by a protease selected from the group consisting ofalanine carboxypeptidase, Armillaria mellea astacin, bacterial leucylaminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosolalanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase,gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase,human rhinovirus 3C protease, hypodermin C, IgA-specific serineendopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC,lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionylaminopeptidase, myxobacter, nardilysin, pancreatic endopeptidase E,picornain 2A, picornain 3C, proendopeptidase, prolyl aminopeptidase,proprotein convertase I, proprotein convertase II, russellysin,saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissuekallikrein, tobacco etch virus (TEV), togavirin, tryptophanylaminopeptidase, U-plasminogen activator, V8, venombin A, venombin AB,and Xaa-pro aminopeptidase.

For example, the proteolytically cleavable linker can comprise a matrixmetalloproteinase cleavage site, e.g., a cleavage site for a MMPselected from collagenase-1, -2, and -3 (MMP-1, -8, and -13), gelatinaseA and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11),matrilysin (MMP-7), and membrane metalloproteinases (MT1-MMP andMT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-X-X-Hy(wherein, X represents an arbitrary residue; Hy, a hydrophobic residue;SEQ ID NO:70), e.g., Pro-X-X-Hy-(Ser/Thr) SEQ ID NO:71, e.g.,Pro-Leu/Gln-Gly-Met-Thr-Ser (SEQ ID NO:72) or Pro-Leu/Gln-Gly-Met-Thr(SEQ ID NO:73). Another example of a protease cleavage site is aplasminogen activator cleavage site, e.g., a uPA or a tissue plasminogenactivator (tPA) cleavage site. Another example of a suitable proteasecleavage site is a prolactin cleavage site. Specific examples ofcleavage sequences of uPA and tPA include sequences comprisingVal-Gly-Arg. Another example of a protease cleavage site that can beincluded in a proteolytically cleavable linker is a tobacco etch virus(TEV) protease cleavage site, e.g., ENLYTQS (SEQ ID NO:6), where theprotease cleaves between the glutamine and the serine. Another exampleof a protease cleavage site that can be included in a proteolyticallycleavable linker is an enterokinase cleavage site, e.g., DDDDK (SEQ IDNO:7), where cleavage occurs after the lysine residue. Another exampleof a protease cleavage site that can be included in a proteolyticallycleavable linker is a thrombin cleavage site, e.g., LVPR (SEQ ID NO:8).Additional suitable linkers comprising protease cleavage sites includelinkers comprising one or more of the following amino acid sequences:LEVLFQGP (SEQ ID NO:5), cleaved by PreScission protease (a fusionprotein comprising human rhinovirus 3C protease andglutathione-S-transferase; Walker et al. (1994) Biotechnol. 12:601); athrombin cleavage site, e.g., CGLVPAGSGP (SEQ ID NO:30); SLLKSRMVPNFN(SEQ ID NO:31) or SLLIARRMPNFN (SEQ ID NO:32), cleaved by cathepsin B;SKLVQASASGVN (SEQ ID NO:33) or SSYLKASDAPDN (SEQ ID NO:34), cleaved byan Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ ID NO:35) cleaved byMMP-3 (stromelysin); SLRPLALWRSFN (SEQ ID NO:36) cleaved by MMP-7(matrilysin); SPQGIAGQRNFN (SEQ ID NO:37) cleaved by MMP-9;DVDERDVRGFASFL SEQ ID NO:38) cleaved by a thermolysin-like MMP;SLPLGLWAPNFN (SEQ ID NO:39) cleaved by matrix metalloproteinase 2(MMP-2); SLLIFRSWANFN (SEQ ID NO:40) cleaved by cathespin L;SGVVIATVIVIT (SEQ ID NO:41) cleaved by cathepsin D; SLGPQGIWGQFN (SEQ IDNO:42) cleaved by matrix metalloproteinase 1 (MMP-1); KKSPGRVVGGSV (SEQID NO:43) cleaved by urokinase-type plasminogen activator; PQGLLGAPGILG(SEQ ID NO:44) cleaved by membrane type 1 matrixmetalloproteinase(MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQ ID NO:45) cleaved bystromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase andstromelysin-1; GPQGLAGQRGIV (SEQ ID NO:46) cleaved by matrixmetalloproteinase 13 (collagenase-3); GGSGQRGRKALE (SEQ ID NO:47)cleaved by tissue-type plasminogen activator (tPA); SLSALLSSDIFN (SEQ IDNO:48) cleaved by human prostate-specific antigen; SLPRFKIIGGFN (SEQ IDNO:49) cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ ID NO:50) cleavedby neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO:51) cleaved bycalpain (calcium activated neutral protease).

The Fc region can be a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc,a human IgG4 Fc, etc. In some cases, the Fc region comprises an aminoacid sequence having having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to an amino acid sequence of an Fc region depicted inFIG. 9A-9C. In some cases, the Fc region comprises an amino acidsequence having having at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the human IgG1 Fc polypeptide depicted in FIG. 9A. In somecases, the Fc region comprises an amino acid sequence having having atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to the human IgG2Fc polypeptide depicted in FIG. 9A; e.g., the Fc region comprises anamino acid sequence having having at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 99-325 of the human IgG2 Fcpolypeptide depicted in FIG. 9A. In some cases, the Fc region comprisesan amino acid sequence having having at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the human IgG3 Fc polypeptide depicted in FIG.9A; e.g., the Fc region comprises an amino acid sequence having havingat least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to amino acids19-246 of the human IgG3 Fc polypeptide depicted in FIG. 9A.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding a fusion protein comprising, in order fromN-terminus to C-terminus: a) an Ig Fc region; b) a proteolyticallycleavable linker comprising the amino acid sequence LEVLFQGP (SEQ IDNO:5) and having a length of from 8 amino acids to 15 amino acids; andc) a variant E2 polypeptide of the present disclosure, where the variantHCV E2 polypeptide comprises: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E2 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E2 polypeptide. In some cases, thenucleotide sequence encoding the fusion protein is operably linked to atranscription control element, e.g., a promoter that is functional in aeukaryotic cell. Suitable promoters include, e.g., a CMV promoter, anSV40 promoter, and the like. In some cases, the nucleic acid is presentin an expression vector (e.g., a lentivirus vector; an adenoassociatedvirus vector; an adenovirus vector; a retroviral vector; a non-viralvector; etc.).

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding a fusion protein comprising, in order fromN-terminus to C-terminus: a) an Ig Fc region; b) a proteolyticallycleavable linker comprising the amino acid sequence ENLYTQS (SEQ IDNO:6) and having a length of from 7 amino acids to 12 amino acids; andc) a variant E2 polypeptide of the present disclosure, where the variantHCV E2 polypeptide comprises: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E2 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E2 polypeptide. In some cases, thenucleotide sequence encoding the fusion protein is operably linked to atranscription control element, e.g., a promoter that is functional in aeukaryotic cell. Suitable promoters include, e.g., a CMV promoter, anSV40 promoter, and the like. In some cases, the nucleic acid is presentin an expression vector (e.g., a lentivirus vector; an adenoassociatedvirus vector; an adenovirus vector; a retroviral vector; a non-viralvector; etc.).

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) an HCV E1 polypeptide; and b) avariant E2 polypeptide of the present disclosure, where the variant HCVE2 polypeptide comprises: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E2 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E2 polypeptide. In some cases, thenucleotide sequence encoding the E1/E2 polypeptide is operably linked toa transcription control element, e.g., a promoter that is functional ina eukaryotic cell. Suitable promoters include, e.g., a CMV promoter, anSV40 promoter, and the like. In some cases, the nucleic acid is presentin an expression vector (e.g., a lentivirus vector; an adenoassociatedvirus vector; an adenovirus vector; a retroviral vector; a non-viralvector; etc.). In some cases, the E1/E2 polypeptide comprises ahost-derived signal peptidase cleavage site between the HCV E1polypeptide and the variant HCV E2 polypeptide. Thus, in some cases, anucleic acid of the present disclosure comprises a nucleotide sequenceencoding an E1/E2 polypeptide comprising, in order from N-terminus toC-terminus: a) an HCV E1 polypeptide; b) a host signal peptidasecleavage site; and c) a variant E2 polypeptide of the presentdisclosure, where the variant HCV E2 polypeptide comprises: i) an HCV E2polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E2 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E2 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E2 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E2polypeptide.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) an HCV E1 polypeptide; b) an Ig Fcregion; and c) a variant E2 polypeptide of the present disclosure, wherethe variant HCV E2 polypeptide comprises: i) an HCV E2 polypeptide; andii) a heterologous polypeptide that comprises one or more T-cellepitopes (e.g., one or more T cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide).In some cases, the variant HCV E2 polypeptide comprises, in order fromN-terminus to C-terminus: i) the HCV E2 polypeptide; and ii) theheterologous polypeptide comprising the one or more T cell epitopes. Inother cases, the variant HCV E2 polypeptide comprises, in order fromN-terminus to C-terminus: i) the heterologous polypeptide comprising theone or more T cell epitopes; and ii) the HCV E2 polypeptide. In somecases, the nucleotide sequence encoding the E1/E2 polypeptide isoperably linked to a transcription control element, e.g., a promoterthat is functional in a eukaryotic cell. Suitable promoters include,e.g., a CMV promoter, an SV40 promoter, and the like. In some cases, thenucleic acid is present in an expression vector (e.g., a lentivirusvector; an adenoassociated virus vector; an adenovirus vector; aretroviral vector; a non-viral vector; etc.). In some cases, the E1/E2polypeptide comprises a host-derived signal peptidase cleavage sitebetween the E1 polypeptide and the Ig Fc region. Thus, in some cases, anucleic acid of the present disclosure comprises a nucleotide sequenceencoding an E1/E2 polypeptide comprising, in order from N-terminus toC-terminus: a) an HCV E1 polypeptide; b) a host-derived signal peptidasecleavage site; c) an Ig Fc region; and d) a variant E2 polypeptide ofthe present disclosure, where the variant HCV E2 polypeptide comprises:i) an HCV E2 polypeptide; and ii) a heterologous polypeptide thatcomprises one or more T-cell epitopes (e.g., one or more T cell epitopespresent in an HCV polypeptide other than an HCV E1 polypeptide or an HCVE2 polypeptide). In some cases, the variant HCV E2 polypeptidecomprises, in order from N-terminus to C-terminus: i) the HCV E2polypeptide; and ii) the heterologous polypeptide comprising the one ormore T cell epitopes. In other cases, the variant HCV E2 polypeptidecomprises, in order from N-terminus to C-terminus: i) the heterologouspolypeptide comprising the one or more T cell epitopes; and ii) the HCVE2 polypeptide. In some cases, a nucleotide sequence encoding from 2 to10 amino acids (e.g., 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or10 aa) of the N-terminus of an E2 polypeptide is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. Examples are depicted in FIG. 5A. For example, in some cases,a nucleotide sequence encoding QT, ET, HT, GT, TT, RH, NT, AY, VI, or STis interposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding ET (Glu-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding QT (Gln-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding TT (Thr-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding GT (Gly-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding HT (His-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding NT (Asn-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding VT (Val-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding ST (Ser-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence.

In some Fc tagged E1E2 constructs (with or without TP insertion), theduplication of the first two amino acids of E2 is such that an aminoacid is created at the N-terminus of E2 following processing by signalpeptidase (SP) (FIG. 5A). Such amino acids at the amino terminus includeasparagine (N), glutamine (Q) or cysteine (C). Such amino acids cantarget the protein for proteasome-mediated degradation via the N-endrule pathway (reviewed in: Tasaki T et al. 2012. Annu Rev Biochem81261-289). For example in FIG. 5A-5B: the insertion of QT in Avi1a129(1A) creates an N-terminal glutamine (Q) residue following cleavage bysignal peptidase. In this case, an alternative amino acid could beselected according to either the consensus sequence for the particulargenotype or a particular genotype subclass (eg: genotype 1A in thiscase). The final purified E1E2 protein containing an N-terminal polytopeaddition (TPx) from Fc tagged E1E2 constructs contains an N-terminalglycine (G) residue and is not expected to be a substrate for the N-endrule pathway.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) an HCV E1 polypeptide; b) an Ig Fcregion; c) a proteolytically cleavable linker; and d) a variant E2polypeptide of the present disclosure, where the variant HCV E2polypeptide comprises: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide). In some cases, the variant HCV E2polypeptide comprises, in order from N-terminus to C-terminus: i) theHCV E2 polypeptide; and ii) the heterologous polypeptide comprising theone or more T cell epitopes. In other cases, the variant HCV E2polypeptide comprises, in order from N-terminus to C-terminus: i) theheterologous polypeptide comprising the one or more T cell epitopes; andii) the HCV E2 polypeptide. In some cases, the nucleotide sequenceencoding the fusion protein is operably linked to a transcriptioncontrol element, e.g., a promoter that is functional in a eukaryoticcell. Suitable promoters include, e.g., a CMV promoter, an SV40promoter, and the like. In some cases, the nucleic acid is present in anexpression vector (e.g., a lentivirus vector; an adenoassociated virusvector; an adenovirus vector; a retroviral vector; a non-viral vector;etc.). In some cases, the E1/E2 polypeptide comprises a host-derivedsignal peptidase cleavage site between the E1 polypeptide and the Ig Fcregion. Thus, in some cases, a nucleic acid of the present disclosurecomprises a nucleotide sequence encoding an E1/E2 polypeptidecomprising, in order from N-terminus to C-terminus: a) an HCV E1polypeptide; b) a host-derived signal peptidase cleavage site; c) an IgFc region; d) a proteolytically cleavable linker; and e) a variant E2polypeptide of the present disclosure, where the variant HCV E2polypeptide comprises: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide). In some cases, the variant HCV E2polypeptide comprises, in order from N-terminus to C-terminus: i) theHCV E2 polypeptide; and ii) the heterologous polypeptide comprising theone or more T cell epitopes. In other cases, the variant HCV E2polypeptide comprises, in order from N-terminus to C-terminus: i) theheterologous polypeptide comprising the one or more T cell epitopes; andii) the HCV E2 polypeptide. In some cases, a nucleotide sequenceencoding from 2 to 10 amino acids (e.g., 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7aa, 8 aa, 9 aa, or 10 aa) of the N-terminus of an E2 polypeptide isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. Examples are depicted in FIG. 5A. Forexample, in some cases, a nucleotide sequence encoding QT, ET, HT, GT,TT, RH, NT, AY, VI, or ST is interposed between the E1-encodingnucleotide sequence and the Ig Fc-encoding nucleotide sequence. In somecases, a nucleotide sequence encoding ET (Glu-Thr) is interposed betweenthe E1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding QT (Gln-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding TT (Thr-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding GT (Gly-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding HT (His-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding NT (Asn-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding VT (Val-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding ST (Ser-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) an HCV E1 polypeptide; b) an Ig Fcregion; c) a proteolytically cleavable linker comprising the amino acidsequence LEVLFQGP (SEQ ID NO:5) and having a length of from 8 aminoacids to 15 amino acids; and d) a variant E2 polypeptide of the presentdisclosure, where the variant HCV E2 polypeptide comprises: i) an HCV E2polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E2 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E2 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E2 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E2polypeptide. In some cases, the nucleotide sequence encoding the fusionprotein is operably linked to a transcription control element, e.g., apromoter that is functional in a eukaryotic cell. Suitable promotersinclude, e.g., a CMV promoter, an SV40 promoter, and the like. In somecases, the nucleic acid is present in an expression vector (e.g., alentivirus vector; an adenoassociated virus vector; an adenovirusvector; a retroviral vector; a non-viral vector; etc.). In some cases,the E1/E2 polypeptide comprises a host-derived signal peptidase cleavagesite between the E1 polypeptide and the Ig Fc region. Thus, in somecases, a nucleic acid of the present disclosure comprises a nucleotidesequence encoding an E1/E2 polypeptide comprising, in order fromN-terminus to C-terminus: a) an HCV E1 polypeptide; b) a host-derivedsignal peptidase cleavage site; c) an Ig Fc region; d) a proteolyticallycleavable linker comprising the amino acid sequence LEVLFQGP (SEQ IDNO:5) and having a length of from 8 amino acids to 15 amino acids; ande) a variant E2 polypeptide of the present disclosure, where the variantHCV E2 polypeptide comprises: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E2 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E2 polypeptide. In some cases, anucleotide sequence encoding from 2 to 10 amino acids (e.g., 2 aa, 3 aa,4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa) of the N-terminus of an E2polypeptide is interposed between the E1-encoding nucleotide sequenceand the Ig Fc-encoding nucleotide sequence. Examples are depicted inFIG. 5A. For example, in some cases, a nucleotide sequence encoding QT,ET, HT, GT, TT, RH, NT, AY, VI, or ST is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding ET (Glu-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding QT (Gln-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding TT (Thr-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding GT (Gly-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding HT (His-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding NT (Asn-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding VT (Val-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding ST (Ser-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) an HCV E1 polypeptide; b) an Ig Fcregion; c) a proteolytically cleavable linker comprising the amino acidsequence ENLYTQS (SEQ ID NO:6) and having a length of from 7 amino acidsto 12 amino acids; and d) a variant E2 polypeptide of the presentdisclosure, where the variant HCV E2 polypeptide comprises: i) an HCV E2polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E2 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E2 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E2 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E2polypeptide. In some cases, the nucleotide sequence encoding the fusionprotein is operably linked to a transcription control element, e.g., apromoter that is functional in a eukaryotic cell. Suitable promotersinclude, e.g., a CMV promoter, an SV40 promoter, and the like. In somecases, the nucleic acid is present in an expression vector (e.g., alentivirus vector; an adenoassociated virus vector; an adenovirusvector; a retroviral vector; a non-viral vector; etc.). In some cases,the E1/E2 polypeptide comprises a host-derived signal peptidase cleavagesite between the E1 polypeptide and the Ig Fc region. Thus, in somecases, a nucleic acid of the present disclosure comprises a nucleotidesequence encoding an E1/E2 polypeptide comprising, in order fromN-terminus to C-terminus: a) an HCV E1 polypeptide; b) a host-derivedsignal peptidase cleavage site; c) an Ig Fc region; d) a proteolyticallycleavable linker comprising the amino acid sequence ENLYTQS (SEQ IDNO:6) and having a length of from 7 amino acids to 12 amino acids; ande) a variant E2 polypeptide of the present disclosure, where the variantHCV E2 polypeptide comprises: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E2 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E2 polypeptide. In some cases, anucleotide sequence encoding from 2 to 10 amino acids (e.g., 2 aa, 3 aa,4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa) of the N-terminus of an E2polypeptide is interposed between the E1-encoding nucleotide sequenceand the Ig Fc-encoding nucleotide sequence. Examples are depicted inFIG. 5A. For example, in some cases, a nucleotide sequence encoding QT,ET, HT, GT, TT, RH, NT, AY, VI, or ST is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding ET (Glu-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding QT (Gln-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding TT (Thr-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding GT (Gly-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding HT (His-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding NT (Asn-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding VT (Val-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding ST (Ser-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) a signal peptide that directs theE1/E2 polypeptide to the endoplasmic reticulum (ER) followingtranslation of the E1/E2 polypeptide; b) an HCV E1 polypeptide; c) an IgFc region; d) a proteolytically cleavable linker; and e) a variant E2polypeptide of the present disclosure, where the variant HCV E2polypeptide comprises: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide). In some cases, the variant HCV E2polypeptide comprises, in order from N-terminus to C-terminus: i) theHCV E2 polypeptide; and ii) the heterologous polypeptide comprising theone or more T cell epitopes. In other cases, the variant HCV E2polypeptide comprises, in order from N-terminus to C-terminus: i) theheterologous polypeptide comprising the one or more T cell epitopes; andii) the HCV E2 polypeptide. In some cases, the nucleotide sequenceencoding the fusion protein is operably linked to a transcriptioncontrol element, e.g., a promoter that is functional in a eukaryoticcell. Suitable promoters include, e.g., a CMV promoter, an SV40promoter, and the like. In some cases, the nucleic acid is present in anexpression vector (e.g., a lentivirus vector; an adenoassociated virusvector; an adenovirus vector; a retroviral vector; a non-viral vector;etc.). In some cases, the E1/E2 polypeptide comprises a host-derivedsignal peptidase cleavage site between the E1 polypeptide and the Ig Fcregion, and comprises a host-derived signal peptidase cleavage sitebetween the signal peptide and the HCV E1 polypeptide. Thus, in somecases, a nucleic acid of the present disclosure comprises a nucleotidesequence encoding an E1/E2 polypeptide comprising, in order fromN-terminus to C-terminus: a) a signal peptide that directs the E1/E2polypeptide to the ER following translation of the E1/E2 polypeptide; b)a host-derived signal peptidase cleavage site; c) an HCV E1 polypeptide;d) a host-derived signal peptidase cleavage site; e) an Ig Fc region; f)a proteolytically cleavable linker; and g) a variant E2 polypeptide ofthe present disclosure, where the variant HCV E2 polypeptide comprises:i) an HCV E2 polypeptide; and ii) a heterologous polypeptide thatcomprises one or more T-cell epitopes (e.g., one or more T cell epitopespresent in an HCV polypeptide other than an HCV E1 polypeptide or an HCVE2 polypeptide). In some cases, the variant HCV E2 polypeptidecomprises, in order from N-terminus to C-terminus: i) the HCV E2polypeptide; and ii) the heterologous polypeptide comprising the one ormore T cell epitopes. In other cases, the variant HCV E2 polypeptidecomprises, in order from N-terminus to C-terminus: i) the heterologouspolypeptide comprising the one or more T cell epitopes; and ii) the HCVE2 polypeptide. In some cases, a nucleotide sequence encoding from 2 to10 amino acids (e.g., 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or10 aa) of the N-terminus of an E2 polypeptide is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. Examples are depicted in FIG. 5A. For example, in some cases,a nucleotide sequence encoding QT, ET, HT, GT, TT, RH, NT, AY, VI, or STis interposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding ET (Glu-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding QT (Gln-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding TT (Thr-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding GT (Gly-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding HT (His-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding NT (Asn-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding VT (Val-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding ST (Ser-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence.

Suitable signal peptides include, e.g., a tissue plasminogen activator(tPA) signal peptide; an Ig kappa light chain precursor signal peptide;a serum albumin preproprotein signal peptide; an Immunoglobulin heavychain signal peptide; an Immunoglobulin light chain signal peptide; anazuorcidin preproprotein signal peptide; a cystatin-S precursor signalpeptide; a trypsinogen-2 precursor signal peptide; a chymotrypsinogenprecursor signal peptide; and the like. (Bendtsen et al. (2004) J. Mol.Biol. 340 783-795; Kober et al. (2012) Biotechnology and Bioengineering110(4) 1164-1173).

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) a signal peptide that directs theE1/E2 polypeptide to the endoplasmic reticulum (ER) followingtranslation of the E1/E2 polypeptide; b) an HCV E1 polypeptide; c) an IgFc region; d) a proteolytically cleavable linker comprising the aminoacid sequence LEVLFQGP (SEQ ID NO:5) and having a length of from 8 aminoacids to 15 amino acids; and e) a variant E2 polypeptide of the presentdisclosure, where the variant HCV E2 polypeptide comprises: i) an HCV E2polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E2 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E2 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E2 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E2polypeptide. In some cases, the nucleotide sequence encoding the fusionprotein is operably linked to a transcription control element, e.g., apromoter that is functional in a eukaryotic cell. Suitable promotersinclude, e.g., a CMV promoter, an SV40 promoter, and the like. In somecases, the nucleic acid is present in an expression vector (e.g., alentivirus vector; an adenoassociated virus vector; an adenovirusvector; a retroviral vector; a non-viral vector; etc.). In some cases,the E1/E2 polypeptide comprises a host-derived signal peptidase cleavagesite between the E1 polypeptide and the Ig Fc region, and comprises ahost-derived signal peptidase cleavage site between the signal peptideand the HCV E1 polypeptide. Thus, in some cases, a nucleic acid of thepresent disclosure comprises a nucleotide sequence encoding an E1/E2polypeptide comprising, in order from N-terminus to C-terminus: a) asignal peptide that directs the E1/E2 polypeptide to the endoplasmicreticulum (ER) following translation of the E1/E2 polypeptide; b) ahost-derived signal peptidase cleavage site; c) an HCV E1 polypeptide;d) a host-derived signal peptidase cleavage site; e) an Ig Fc region; f)a proteolytically cleavable linker comprising the amino acid sequenceLEVLFQGP (SEQ ID NO:5) and having a length of from 8 amino acids to 15amino acids; and g) a variant E2 polypeptide of the present disclosure,where the variant HCV E2 polypeptide comprises: i) an HCV E2polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E2 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E2 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E2 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E2polypeptide. In some cases, a nucleotide sequence encoding from 2 to 10amino acids (e.g., 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10aa) of the N-terminus of an E2 polypeptide is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. Examples are depicted in FIG. 5A. For example, in some cases,a nucleotide sequence encoding QT, ET, HT, GT, TT, RH, NT, AY, VI, or STis interposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding ET (Glu-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding QT (Gln-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding TT (Thr-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding GT (Gly-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding HT (His-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding NT (Asn-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding VT (Val-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding ST (Ser-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) a signal peptide that directs theE1/E2 polypeptide to the endoplasmic reticulum (ER) followingtranslation of the E1/E2 polypeptide; b) an HCV E1 polypeptide; c) an IgFc region; d) a proteolytically cleavable linker comprising the aminoacid sequence ENLYTQS (SEQ ID NO:6) and having a length of from 7 aminoacids to 12 amino acids; and e) a variant E2 polypeptide of the presentdisclosure, where the variant HCV E2 polypeptide comprises: i) an HCV E2polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E2 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E2 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E2 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E2polypeptide. In some cases, the nucleotide sequence encoding the fusionprotein is operably linked to a transcription control element, e.g., apromoter that is functional in a eukaryotic cell. Suitable promotersinclude, e.g., a CMV promoter, an SV40 promoter, and the like. In somecases, the nucleic acid is present in an expression vector (e.g., alentivirus vector; an adenoassociated virus vector; an adenovirusvector; a retroviral vector; a non-viral vector; etc.). In some cases,the E1/E2 polypeptide comprises a host-derived signal peptidase cleavagesite between the E1 polypeptide and the Ig Fc region, and comprises ahost-derived signal peptidase cleavage site between the signal peptideand the HCV E1 polypeptide. Thus, in some cases, a nucleic acid of thepresent disclosure comprises a nucleotide sequence encoding an E1/E2polypeptide comprising, in order from N-terminus to C-terminus: a) asignal peptide that directs the E1/E2 polypeptide to the ER followingtranslation of the E1/E2 polypeptide; b) a host-derived signal peptidasecleavage site; c) an HCV E1 polypeptide; d) a host-derived signalpeptidase cleavage site; e) an Ig Fc region; f) a proteolyticallycleavable linker comprising the amino acid sequence ENLYTQS (SEQ IDNO:6) and having a length of from 7 amino acids to 12 amino acids; andg) a variant E2 polypeptide of the present disclosure, where the variantHCV E2 polypeptide comprises: i) an HCV E2 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E2 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E2 polypeptide. In some cases, anucleotide sequence encoding from 2 to 10 amino acids (e.g., 2 aa, 3 aa,4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa) of the N-terminus of an E2polypeptide is interposed between the E1-encoding nucleotide sequenceand the Ig Fc-encoding nucleotide sequence. Examples are depicted inFIG. 5A. For example, in some cases, a nucleotide sequence encoding QT,ET, HT, GT, TT, RH, NT, AY, VI, or ST is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding ET (Glu-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding QT (Gln-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding TT (Thr-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding GT (Gly-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding HT (His-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence. In some cases, anucleotide sequence encoding NT (Asn-Thr) is interposed between theE1-encoding nucleotide sequence and the Ig Fc-encoding nucleotidesequence. In some cases, a nucleotide sequence encoding VT (Val-Thr) isinterposed between the E1-encoding nucleotide sequence and the IgFc-encoding nucleotide sequence. In some cases, a nucleotide sequenceencoding ST (Ser-Thr) is interposed between the E1-encoding nucleotidesequence and the Ig Fc-encoding nucleotide sequence.

As noted above, in some cases, a nucleic acid of the present disclosureis present in an expression vector. Thus, the present disclosureprovides a recombinant expression vector comprising a nucleic acidcomprising a nucleotide sequence encoding a variant E2 polypeptide ofthe present disclosure or comprising a nucleotide sequence encoding anE1/E2 heterodimer of the present disclosure. In some cases, thenucleotide sequence encoding the variant E2 polypeptide of the presentdisclosure or the nucleotide sequence encoding the E1/E2 heterodimer ofthe present disclosure is operably linked to a transcriptional controlelement, e.g., a promoter, such as a promoter functional in a eukaryoticcell.

Suitable expression vectors include, but are not limited to, baculovirusvectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids,bacterial artificial chromosomes, viral vectors (e.g. viral vectorsbased on vaccinia virus, poliovirus, adenovirus, adeno-associated virus,SV40, herpes simplex virus, HIV-based lentivirus vectors, murineleukemia virus (MVL)-based gamma retrovirus vectors, and the like),P1-based artificial chromosomes, yeast plasmids, yeast artificialchromosomes, and any other vectors specific for specific hosts ofinterest (such as E. coli, mammalian cells, insect cells, or yeastcells).

IV(B). Variant E1 and E1/E2 Heterodimers Comprising a Variant E1Polypeptide

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a variant HCV E1 polypeptide of the presentdisclosure, where the variant HCV E1 polypeptide comprises: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). The present disclosure provides a nucleic acid comprisinga nucleotide sequence encoding a polyprotein comprising a variant E1polypeptide of the present disclosure. In some cases, the nucleic acidis present in an expression vector. Thus, the present disclosureprovides a recombinant expression vector comprising a nucleotidesequence encoding a variant E1 polypeptide of the present disclosure,where the variant HCV E1 polypeptide comprises: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide).

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding a variant E1 polypeptide of the presentdisclosure, where the variant HCV E1 polypeptide comprises: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the nucleotide sequence encoding thevariant E2 polypeptide is operably linked to a transcription controlelement, e.g., a promoter that is functional in a eukaryotic cell.Suitable promoters include, e.g., a cytomegalovirus (CMV) promoter, anSV40 promoter, and the like. In some cases, the nucleic acid is presentin an expression vector (e.g., a lentivirus vector; an adenoassociatedvirus vector; an adenovirus vector; a retroviral vector; a non-viralvector; etc.). In some cases, the nucleic acid is present in anexpression vector, where the expression vector is a non-viral vector.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding a fusion protein comprising, in order fromN-terminus to C-terminus: a) an immunoglobulin (Ig) Fc region; and b) avariant E1 polypeptide of the present disclosure, where the variant HCVE1 polypeptide comprises: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E1 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In some cases,the variant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E1 polypeptide. In some cases, thenucleotide sequence encoding the fusion protein is operably linked to atranscription control element, e.g., a promoter that is functional in aeukaryotic cell. Suitable promoters include, e.g., a CMV promoter, anSV40 promoter, and the like. In some cases, the nucleic acid is presentin an expression vector (e.g., a lentivirus vector; an adenoassociatedvirus vector; an adenovirus vector; a retroviral vector; a non-viralvector; etc.).

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding a fusion protein comprising, in order fromN-terminus to C-terminus: a) an Ig Fc region; b) a proteolyticallycleavable linker; and c) a variant E1 polypeptide of the presentdisclosure, where the variant HCV E1 polypeptide comprises: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E1 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E1 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E1 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E1polypeptide. In some cases, the nucleotide sequence encoding the fusionprotein is operably linked to a transcription control element, e.g., apromoter that is functional in a eukaryotic cell. Suitable promotersinclude, e.g., a CMV promoter, an SV40 promoter, and the like. In somecases, the nucleic acid is present in an expression vector (e.g., alentivirus vector; an adenoassociated virus vector; an adenovirusvector; a retroviral vector; a non-viral vector; etc.).

The proteolytically cleavable linker can include a protease recognitionsequence recognized by a protease selected from the group consisting ofalanine carboxypeptidase, Armillaria mellea astacin, bacterial leucylaminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosolalanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase,gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase,human rhinovirus 3C protease, hypodermin C, IgA-specific serineendopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC,lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionylaminopeptidase, myxobacter, nardilysin, pancreatic endopeptidase E,picornain 2A, picornain 3C, proendopeptidase, prolyl aminopeptidase,proprotein convertase I, proprotein convertase II, russellysin,saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissuekallikrein, tobacco etch virus (TEV), togavirin, tryptophanylaminopeptidase, U-plasminogen activator, V8, venombin A, venombin AB,and Xaa-pro aminopeptidase.

For example, the proteolytically cleavable linker can comprise a matrixmetalloproteinase cleavage site, e.g., a cleavage site for a MMPselected from collagenase-1, -2, and -3 (MMP-1, -8, and -13), gelatinaseA and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11),matrilysin (MMP-7), and membrane metalloproteinases (MT1-MMP andMT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-X-X-Hy(wherein, X represents an arbitrary residue; Hy, a hydrophobic residue;SEQ ID NO:70), e.g., Pro-X-X-Hy-(Ser/Thr) SEQ ID NO:71, e.g.,Pro-Leu/Gln-Gly-Met-Thr-Ser (SEQ ID NO:72) or Pro-Leu/Gln-Gly-Met-Thr(SEQ ID NO:73). Another example of a protease cleavage site is aplasminogen activator cleavage site, e.g., a uPA or a tissue plasminogenactivator (tPA) cleavage site. In some cases, the cleavage site isafurin cleavage site. Specific examples of cleavage sequences of uPA andtPA include sequences comprising Val-Gly-Arg. Another example of aprotease cleavage site that can be included in a proteolyticallycleavable linker is a tobacco etch virus (TEV) protease cleavage site,e.g., ENLYTQS (SEQ ID NO:6), where the protease cleaves between theglutamine and the serine. Another example of a protease cleavage sitethat can be included in a proteolytically cleavable linker is anenterokinase cleavage site, e.g., DDDDK (SEQ ID NO:7), where cleavageoccurs after the lysine residue. Another example of a protease cleavagesite that can be included in a proteolytically cleavable linker is athrombin cleavage site, e.g., LVPR (SEQ ID NO:8). Additional suitablelinkers comprising protease cleavage sites include linkers comprisingone or more of the following amino acid sequences: LEVLFQGP (SEQ IDNO:5), cleaved by PreScission protease (a fusion protein comprisinghuman rhinovirus 3C protease and glutathione-S-transferase; Walker etal. (1994) Biotechnol. 12:601); a thrombin cleavage site, e.g.,CGLVPAGSGP (SEQ ID NO:30); SLLKSRMVPNFN (SEQ ID NO:31) or SLLIARRMPNFN(SEQ ID NO:32), cleaved by cathepsin B; SKLVQASASGVN (SEQ ID NO:33) orSSYLKASDAPDN (SEQ ID NO:34), cleaved by an Epstein-Barr virus protease;RPKPQQFFGLMN (SEQ ID NO:35) cleaved by MMP-3 (stromelysin); SLRPLALWRSFN(SEQ ID NO:36) cleaved by MMP-7 (matrilysin); SPQGIAGQRNFN (SEQ IDNO:37) cleaved by MMP-9; DVDERDVRGFASFL SEQ ID NO:38) cleaved by athermolysin-like MMP; SLPLGLWAPNFN (SEQ ID NO:39) cleaved by matrixmetalloproteinase 2 (MMP-2); SLLIFRSWANFN (SEQ ID NO:40) cleaved bycathespin L; SGVVIATVIVIT (SEQ ID NO:41) cleaved by cathepsin D;SLGPQGIWGQFN (SEQ ID NO:42) cleaved by matrix metalloproteinase 1(MMP-1); KKSPGRVVGGSV (SEQ ID NO:43) cleaved by urokinase-typeplasminogen activator; PQGLLGAPGILG (SEQ ID NO:44) cleaved by membranetype 1 matrixmetalloproteinase (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT(SEQ ID NO:45) cleaved by stromelysin 3 (or MMP-11), thermolysin,fibroblast collagenase and stromelysin-1; GPQGLAGQRGIV (SEQ ID NO:46)cleaved by matrix metalloproteinase 13 (collagenase-3); GGSGQRGRKALE(SEQ ID NO:47) cleaved by tissue-type plasminogen activator (tPA);SLSALLSSDIFN (SEQ ID NO:48) cleaved by human prostate-specific antigen;SLPRFKIIGGFN (SEQ ID NO:49) cleaved by kallikrein (hK3); SLLGIAVPGNFN(SEQ ID NO:50) cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ IDNO:51) cleaved by calpain (calcium activated neutral protease).

The Fc region can be a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc,a human IgG4 Fc, etc. In some cases, the Fc region comprises an aminoacid sequence having at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an Fc region depicted in FIG.9A-9C. In some cases, the Fc region comprises an amino acid sequencehaving at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thehuman IgG1 Fc polypeptide depicted in FIG. 9A. In some cases, the Fcregion comprises an amino acid sequence having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the human IgG2 Fc polypeptidedepicted in FIG. 9A; e.g., the Fc region comprises an amino acidsequence having at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 99-325 of the human IgG2 Fc polypeptide depictedin FIG. 9A. In some cases, the Fc region comprises an amino acidsequence having at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the human IgG3 Fc polypeptide depicted in FIG. 9A; e.g., theFc region comprises an amino acid sequence having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 19-246 of the humanIgG3 Fc polypeptide depicted in FIG. 9A.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding a fusion protein comprising, in order fromN-terminus to C-terminus: a) an Ig Fc region; b) a proteolyticallycleavable linker comprising the amino acid sequence LEVLFQGP (SEQ IDNO:5) and having a length of from 8 amino acids to 15 amino acids; andc) a variant E1 polypeptide of the present disclosure, where the variantHCV E1 polypeptide comprises: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E1 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E1 polypeptide. In some cases, thenucleotide sequence encoding the fusion protein is operably linked to atranscription control element, e.g., a promoter that is functional in aeukaryotic cell. Suitable promoters include, e.g., a CMV promoter, anSV40 promoter, and the like. In some cases, the nucleic acid is presentin an expression vector (e.g., a lentivirus vector; an adenoassociatedvirus vector; an adenovirus vector; a retroviral vector; a non-viralvector; etc.).

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding a fusion protein comprising, in order fromN-terminus to C-terminus: a) an Ig Fc region; b) a proteolyticallycleavable linker comprising the amino acid sequence ENLYTQS (SEQ IDNO:6) and having a length of from 7 amino acids to 12 amino acids; andc) a variant E1 polypeptide of the present disclosure, where the variantHCV E1 polypeptide comprises: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E1 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E1 polypeptide. In some cases, thenucleotide sequence encoding the fusion protein is operably linked to atranscription control element, e.g., a promoter that is functional in aeukaryotic cell. Suitable promoters include, e.g., a CMV promoter, anSV40 promoter, and the like. In some cases, the nucleic acid is presentin an expression vector (e.g., a lentivirus vector; an adenoassociatedvirus vector; an adenovirus vector; a retroviral vector; a non-viralvector; etc.).

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) an HCV E2 polypeptide; and b) avariant E1 polypeptide of the present disclosure, where the variant HCVE1 polypeptide comprises: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E1 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E1 polypeptide. In some cases, thenucleotide sequence encoding the E1/E2 polypeptide is operably linked toa transcription control element, e.g., a promoter that is functional ina eukaryotic cell. Suitable promoters include, e.g., a CMV promoter, anSV40 promoter, and the like. In some cases, the nucleic acid is presentin an expression vector (e.g., a lentivirus vector; an adenoassociatedvirus vector; an adenovirus vector; a retroviral vector; a non-viralvector; etc.). In some cases, the E1/E2 polypeptide comprises ahost-derived signal peptidase cleavage site between the HCV E2polypeptide and the variant HCV E1 polypeptide. Thus, in some cases, anucleic acid of the present disclosure comprises a nucleotide sequenceencoding an E1/E2 polypeptide comprising, in order from N-terminus toC-terminus: a) an HCV E2 polypeptide; b) a host signal peptidasecleavage site; and c) a variant E1 polypeptide of the presentdisclosure, where the variant HCV E1 polypeptide comprises: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E1 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E1 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E1 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E1polypeptide.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) an HCV E2 polypeptide; b) an Ig Fcregion; and c) a variant E1 polypeptide of the present disclosure, wherethe variant HCV E1 polypeptide comprises: i) an HCV E1 polypeptide; andii) a heterologous polypeptide that comprises one or more T-cellepitopes (e.g., one or more T cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide).In some cases, the variant HCV E1 polypeptide comprises, in order fromN-terminus to C-terminus: i) the HCV E1 polypeptide; and ii) theheterologous polypeptide comprising the one or more T cell epitopes. Inother cases, the variant HCV E1 polypeptide comprises, in order fromN-terminus to C-terminus: i) the heterologous polypeptide comprising theone or more T cell epitopes; and ii) the HCV E1 polypeptide. In somecases, the nucleotide sequence encoding the E1/E2 polypeptide isoperably linked to a transcription control element, e.g., a promoterthat is functional in a eukaryotic cell. Suitable promoters include,e.g., a CMV promoter, an SV40 promoter, and the like. In some cases, thenucleic acid is present in an expression vector (e.g., a lentivirusvector; an adenoassociated virus vector; an adenovirus vector; aretroviral vector; a non-viral vector; etc.). In some cases, the E/E2polypeptide comprises a host-derived signal peptidase cleavage sitebetween the E2 polypeptide and the Ig Fc region. Thus, in some cases, anucleic acid of the present disclosure comprises a nucleotide sequenceencoding an E1/E2 polypeptide comprising, in order from N-terminus toC-terminus: a) an HCV E2 polypeptide; b) a host-derived signal peptidasecleavage site; c) an Ig Fc region; and d) a variant E1 polypeptide ofthe present disclosure, where the variant HCV E1 polypeptide comprises:i) an HCV E1 polypeptide; and ii) a heterologous polypeptide thatcomprises one or more T-cell epitopes (e.g., one or more T cell epitopespresent in an HCV polypeptide other than an HCV E1 polypeptide or an HCVE2 polypeptide). In some cases, the variant HCV E1 polypeptidecomprises, in order from N-terminus to C-terminus: i) the HCV E1polypeptide; and ii) the heterologous polypeptide comprising the one ormore T cell epitopes. In other cases, the variant HCV E1 polypeptidecomprises, in order from N-terminus to C-terminus: i) the heterologouspolypeptide comprising the one or more T cell epitopes; and ii) the HCVE1 polypeptide.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) an HCV E2 polypeptide; b) an Ig Fcregion; c) a proteolytically cleavable linker; and d) a variant E1polypeptide of the present disclosure, where the variant HCV E1polypeptide comprises: i) an HCV E1 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide). In some cases, the variant HCV E1polypeptide comprises, in order from N-terminus to C-terminus: i) theHCV E1 polypeptide; and ii) the heterologous polypeptide comprising theone or more T cell epitopes. In other cases, the variant HCV E1polypeptide comprises, in order from N-terminus to C-terminus: i) theheterologous polypeptide comprising the one or more T cell epitopes; andii) the HCV E1 polypeptide. In some cases, the nucleotide sequenceencoding the fusion protein is operably linked to a transcriptioncontrol element, e.g., a promoter that is functional in a eukaryoticcell. Suitable promoters include, e.g., a CMV promoter, an SV40promoter, and the like. In some cases, the nucleic acid is present in anexpression vector (e.g., a lentivirus vector; an adenoassociated virusvector; an adenovirus vector; a retroviral vector; a non-viral vector;etc.). In some cases, the E1/E2 polypeptide comprises a host-derivedsignal peptidase cleavage site between the E2 polypeptide and the Ig Fcregion. Thus, in some cases, a nucleic acid of the present disclosurecomprises a nucleotide sequence encoding an E1/E2 polypeptidecomprising, in order from N-terminus to C-terminus: a) an HCV E2polypeptide; b) a host-derived signal peptidase cleavage site; c) an IgFc region; d) a proteolytically cleavable linker; and e) a variant E1polypeptide of the present disclosure, where the variant HCV E1polypeptide comprises: i) an HCV E1 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide). In some cases, the variant HCV E1polypeptide comprises, in order from N-terminus to C-terminus: i) theHCV E1 polypeptide; and ii) the heterologous polypeptide comprising theone or more T cell epitopes. In other cases, the variant HCV E1polypeptide comprises, in order from N-terminus to C-terminus: i) theheterologous polypeptide comprising the one or more T cell epitopes; andii) the HCV E1 polypeptide.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) an HCV E2 polypeptide; b) an Ig Fcregion; c) a proteolytically cleavable linker comprising the amino acidsequence LEVLFQGP (SEQ ID NO:5) and having a length of from 8 aminoacids to 15 amino acids; and d) a variant E1 polypeptide of the presentdisclosure, where the variant HCV E1 polypeptide comprises: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E1 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E1 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E1 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E1polypeptide. In some cases, the nucleotide sequence encoding the fusionprotein is operably linked to a transcription control element, e.g., apromoter that is functional in a eukaryotic cell. Suitable promotersinclude, e.g., a CMV promoter, an SV40 promoter, and the like. In somecases, the nucleic acid is present in an expression vector (e.g., alentivirus vector; an adenoassociated virus vector; an adenovirusvector; a retroviral vector; a non-viral vector; etc.). In some cases,the E1/E2 polypeptide comprises a host-derived signal peptidase cleavagesite between the E2 polypeptide and the Ig Fc region. Thus, in somecases, a nucleic acid of the present disclosure comprises a nucleotidesequence encoding an E1/E2 polypeptide comprising, in order fromN-terminus to C-terminus: a) an HCV E2 polypeptide; b) a host-derivedsignal peptidase cleavage site; c) an Ig Fc region; d) a proteolyticallycleavable linker comprising the amino acid sequence LEVLFQGP (SEQ IDNO:5) and having a length of from 8 amino acids to 15 amino acids; ande) a variant E1 polypeptide of the present disclosure, where the variantHCV E1 polypeptide comprises: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E1 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E1 polypeptide.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) an HCV E2 polypeptide; b) an Ig Fcregion; c) a proteolytically cleavable linker comprising the amino acidsequence ENLYTQS (SEQ ID NO:6) and having a length of from 7 amino acidsto 12 amino acids; and d) a variant E1 polypeptide of the presentdisclosure, where the variant HCV E1 polypeptide comprises: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E1 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E1 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E1 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E1polypeptide. In some cases, the nucleotide sequence encoding the fusionprotein is operably linked to a transcription control element, e.g., apromoter that is functional in a eukaryotic cell. Suitable promotersinclude, e.g., a CMV promoter, an SV40 promoter, and the like. In somecases, the nucleic acid is present in an expression vector (e.g., alentivirus vector; an adenoassociated virus vector; an adenovirusvector; a retroviral vector; a non-viral vector; etc.). In some cases,the E1/E2 polypeptide comprises a host-derived signal peptidase cleavagesite between the E1 polypeptide and the Ig Fc region. Thus, in somecases, a nucleic acid of the present disclosure comprises a nucleotidesequence encoding an E1/E2 polypeptide comprising, in order fromN-terminus to C-terminus: a) an HCV E2 polypeptide; b) a host-derivedsignal peptidase cleavage site; c) an Ig Fc region; d) a proteolyticallycleavable linker comprising the amino acid sequence ENLYTQS (SEQ IDNO:6) and having a length of from 7 amino acids to 12 amino acids; ande) a variant E1 polypeptide of the present disclosure, where the variantHCV E1 polypeptide comprises: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E1 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E1 polypeptide.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) a signal peptide that directs theE1/E2 polypeptide to the endoplasmic reticulum (ER) followingtranslation of the E1/E2 polypeptide; b) an HCV E2 polypeptide; c) an IgFc region; d) a proteolytically cleavable linker; and e) a variant E1polypeptide of the present disclosure, where the variant HCV E1polypeptide comprises: i) an HCV E1 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide). In some cases, the variant HCV E1polypeptide comprises, in order from N-terminus to C-terminus: i) theHCV E1 polypeptide; and ii) the heterologous polypeptide comprising theone or more T cell epitopes. In other cases, the variant HCV E1polypeptide comprises, in order from N-terminus to C-terminus: i) theheterologous polypeptide comprising the one or more T cell epitopes; andii) the HCV E1 polypeptide. In some cases, the nucleotide sequenceencoding the fusion protein is operably linked to a transcriptioncontrol element, e.g., a promoter that is functional in a eukaryoticcell. Suitable promoters include, e.g., a CMV promoter, an SV40promoter, and the like. In some cases, the nucleic acid is present in anexpression vector (e.g., a lentivirus vector; an adenoassociated virusvector; an adenovirus vector; a retroviral vector; a non-viral vector;etc.). In some cases, the E1/E2 polypeptide comprises a host-derivedsignal peptidase cleavage site between the E1 polypeptide and the Ig Fcregion, and comprises a host-derived signal peptidase cleavage sitebetween the signal peptide and the HCV E2 polypeptide. Thus, in somecases, a nucleic acid of the present disclosure comprises a nucleotidesequence encoding an E1/E2 polypeptide comprising, in order fromN-terminus to C-terminus: a) a signal peptide that directs the E1/E2polypeptide to the ER following translation of the E1/E2 polypeptide; b)a host-derived signal peptidase cleavage site; c) an HCV E2 polypeptide;d) a host-derived signal peptidase cleavage site; e) an Ig Fc region; f)a proteolytically cleavable linker; and g) a variant E1 polypeptide ofthe present disclosure, where the variant HCV E1 polypeptide comprises:i) an HCV E1 polypeptide; and ii) a heterologous polypeptide thatcomprises one or more T-cell epitopes (e.g., one or more T cell epitopespresent in an HCV polypeptide other than an HCV E1 polypeptide or an HCVE2 polypeptide). In some cases, the variant HCV E1 polypeptidecomprises, in order from N-terminus to C-terminus: i) the HCV E1polypeptide; and ii) the heterologous polypeptide comprising the one ormore T cell epitopes. In other cases, the variant HCV E1 polypeptidecomprises, in order from N-terminus to C-terminus: i) the heterologouspolypeptide comprising the one or more T cell epitopes; and ii) the HCVE1 polypeptide.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) a signal peptide that directs theE1/E2 polypeptide to the endoplasmic reticulum (ER) followingtranslation of the E1/E2 polypeptide; b) an HCV E2 polypeptide; c) an IgFc region; d) a proteolytically cleavable linker comprising the aminoacid sequence LEVLFQGP (SEQ ID NO:5) and having a length of from 8 aminoacids to 15 amino acids; and e) a variant E1 polypeptide of the presentdisclosure, where the variant HCV E1 polypeptide comprises: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E1 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E1 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E1 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E1polypeptide. In some cases, the nucleotide sequence encoding the fusionprotein is operably linked to a transcription control element, e.g., apromoter that is functional in a eukaryotic cell. Suitable promotersinclude, e.g., a CMV promoter, an SV40 promoter, and the like. In somecases, the nucleic acid is present in an expression vector (e.g., alentivirus vector; an adenoassociated virus vector; an adenovirusvector; a retroviral vector; a non-viral vector; etc.). In some cases,the E1/E2 polypeptide comprises a host-derived signal peptidase cleavagesite between the E2 polypeptide and the Ig Fc region, and comprises ahost-derived signal peptidase cleavage site between the signal peptideand the HCV E2 polypeptide. Thus, in some cases, a nucleic acid of thepresent disclosure comprises a nucleotide sequence encoding an E1/E2polypeptide comprising, in order from N-terminus to C-terminus: a) asignal peptide that directs the E1/E2 polypeptide to the endoplasmicreticulum (ER) following translation of the E1/E2 polypeptide; b) ahost-derived signal peptidase cleavage site; c) an HCV E2 polypeptide;d) a host-derived signal peptidase cleavage site; e) an Ig Fc region; f)a proteolytically cleavable linker comprising the amino acid sequenceLEVLFQGP (SEQ ID NO:5) and having a length of from 8 amino acids to 15amino acids; and g) a variant E1 polypeptide of the present disclosure,where the variant HCV E1 polypeptide comprises: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E1 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E1 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E1 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E1polypeptide.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding an E1/E2 polypeptide comprising, in orderfrom N-terminus to C-terminus: a) a signal peptide that directs theE1/E2 polypeptide to the endoplasmic reticulum (ER) followingtranslation of the E1/E2 polypeptide; b) an HCV E2 polypeptide; c) an IgFc region; d) a proteolytically cleavable linker comprising the aminoacid sequence ENLYTQS (SEQ ID NO:6) and having a length of from 7 aminoacids to 12 amino acids; and e) a variant E1 polypeptide of the presentdisclosure, where the variant HCV E1 polypeptide comprises: i) an HCV E1polypeptide; and ii) a heterologous polypeptide that comprises one ormore T-cell epitopes (e.g., one or more T cell epitopes present in anHCV polypeptide other than an HCV E1 polypeptide or an HCV E2polypeptide). In some cases, the variant HCV E1 polypeptide comprises,in order from N-terminus to C-terminus: i) the HCV E1 polypeptide; andii) the heterologous polypeptide comprising the one or more T cellepitopes. In other cases, the variant HCV E1 polypeptide comprises, inorder from N-terminus to C-terminus: i) the heterologous polypeptidecomprising the one or more T cell epitopes; and ii) the HCV E1polypeptide. In some cases, the nucleotide sequence encoding the fusionprotein is operably linked to a transcription control element, e.g., apromoter that is functional in a eukaryotic cell. Suitable promotersinclude, e.g., a CMV promoter, an SV40 promoter, and the like. In somecases, the nucleic acid is present in an expression vector (e.g., alentivirus vector; an adenoassociated virus vector; an adenovirusvector; a retroviral vector; a non-viral vector; etc.). In some cases,the E1/E2 polypeptide comprises a host-derived signal peptidase cleavagesite between the E2 polypeptide and the Ig Fc region, and comprises ahost-derived signal peptidase cleavage site between the signal peptideand the HCV E2 polypeptide. Thus, in some cases, a nucleic acid of thepresent disclosure comprises a nucleotide sequence encoding an E1/E2polypeptide comprising, in order from N-terminus to C-terminus: a) asignal peptide that directs the E1/E2 polypeptide to the ER followingtranslation of the E1/E2 polypeptide; b) a host-derived signal peptidasecleavage site; c) an HCV E2 polypeptide; d) a host-derived signalpeptidase cleavage site; e) an Ig Fc region; f) a proteolyticallycleavable linker comprising the amino acid sequence ENLYTQS (SEQ IDNO:6) and having a length of from 7 amino acids to 12 amino acids; andg) a variant E1 polypeptide of the present disclosure, where the variantHCV E1 polypeptide comprises: i) an HCV E1 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide). In some cases, thevariant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the HCV E1 polypeptide; and ii) the heterologouspolypeptide comprising the one or more T cell epitopes. In other cases,the variant HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: i) the heterologous polypeptide comprising the one or more Tcell epitopes; and ii) the HCV E1 polypeptide.

As noted above, in some cases, a nucleic acid of the present disclosureis present in an expression vector. Thus, the present disclosureprovides a recombinant expression vector comprising a nucleic acidcomprising a nucleotide sequence encoding a variant E1 polypeptide ofthe present disclosure or comprising a nucleotide sequence encoding anE1/E2 heterodimer of the present disclosure. In some cases, thenucleotide sequence encoding the variant E1 polypeptide of the presentdisclosure or the nucleotide sequence encoding the E1/E2 heterodimer ofthe present disclosure is operably linked to a transcriptional controlelement, e.g., a promoter, such as a promoter functional in a eukaryoticcell.

Suitable expression vectors include, but are not limited to, baculovirusvectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids,bacterial artificial chromosomes, viral vectors (e.g. viral vectorsbased on vaccinia virus, poliovirus, adenovirus, adeno-associated virus,SV40, herpes simplex virus, HIV-based lentivirus vectors, murineleukemia virus (MVL)-based gamma retrovirus vectors, and the like),P1-based artificial chromosomes, yeast plasmids, yeast artificialchromosomes, and any other vectors specific for specific hosts ofinterest (such as E. coli, mammalian cells, insect cells, or yeastcells).

ADDITIONAL EMBODIMENTS

In some cases, a nucleic acid of the present disclosure comprises afirst nucleotide sequence encoding a variant E2 polypeptide of thepresent disclosure, where the nucleotide sequence encodes a polypeptidecomprising, from N-terminus to C-terminus: a) a variant HCV E2polypeptide comprising, in order from N-terminus to C-terminus: i) anHCV E2 polypeptide; and ii) one or more T-cell epitopes from an HCVpolypeptide other than E1 or E2; b) a proteolytically cleavable linker;and c) an Ig Fc polypeptide. In some cases, the nucleic acid furtherincludes a second nucleotide sequence 5′ of the first nucleotidesequence, where the second nucleotide sequence encodes an HCV E1polypeptide. In some cases, the nucleic acid further includes a secondnucleotide sequence 5′ of the first nucleotide sequence, where thesecond nucleotide sequence encodes a variant HCV E1 polypeptide, thevariant HCV E1 polypeptide comprising, in order from N-terminus toC-terminus: i) an HCV E1 polypeptide; and ii) one or more T-cellepitopes from an HCV polypeptide other than E1 or E2.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding a variant E1 polypeptide of the presentdisclosure, where the nucleotide sequence encodes a polypeptidecomprising, from N-terminus to C-terminus: a) a variant HCV E1polypeptide comprising, in order from N-terminus to C-terminus: i) anHCV E1 polypeptide; and ii) one or more T-cell epitopes from an HCVpolypeptide other than E1 or E2; b) a proteolytically cleavable linker;and c) an Ig Fc polypeptide. In some cases, the nucleic acid furtherincludes a second nucleotide sequence 5′ of the first nucleotidesequence, where the second nucleotide sequence encodes an HCV E2polypeptide. In some cases, the nucleic acid further includes a secondnucleotide sequence 5′ of the first nucleotide sequence, where thesecond nucleotide sequence encodes a variant HCV E2 polypeptide, thevariant HCV E2 polypeptide comprising, in order from N-terminus toC-terminus: i) an HCV E2 polypeptide; and ii) one or more T-cellepitopes from an HCV polypeptide other than E1 or E2.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding a polypeptide comprising, from N-terminusto C-terminus: a) an Ig Fc polypeptide; b) a proteolytically cleavablelinker; c) an HCV E1 polypeptide; d) two amino acids from the N-terminusof E2; and e) an HCV E2 polypeptide. In some cases, a nucleic acid ofthe present disclosure comprises a nucleotide sequence encoding apolypeptide comprising, from N-terminus to C-terminus: a) an Ig Fcpolypeptide; b) a proteolytically cleavable linker; c) a variant HCV E1polypeptide comprising, in order from N-terminus to C-terminus: i) anHCV E1 polypeptide; and ii) one or more T-cell epitopes from an HCVpolypeptide other than E1 or E2; d) two amino acids from the N-terminusof E2; and e) an HCV E2 polypeptide. In some cases, a nucleic acid ofthe present disclosure comprises a nucleotide sequence encoding apolypeptide comprising, from N-terminus to C-terminus: a) an Ig Fcpolypeptide; b) a proteolytically cleavable linker; c) a variant HCV E1polypeptide comprising, in order from N-terminus to C-terminus: i) oneor more T-cell epitopes from an HCV polypeptide other than E1 or E2; andii) an HCV E1 polypeptide; d) two amino acids from the N-terminus of E2;and e) an HCV E2 polypeptide. In some cases, a nucleic acid of thepresent disclosure comprises a nucleotide sequence encoding apolypeptide comprising, from N-terminus to C-terminus: a) an Ig Fcpolypeptide; b) a proteolytically cleavable linker; c) an HCV E1polypeptide; d) two amino acids from the N-terminus of E2; and e) avariant HCV E2 polypeptide comprising, in order from N-terminus toC-terminus: i) an HCV E2 polypeptide; and ii) one or more T-cellepitopes from an HCV polypeptide other than E1 or E2. In some cases, anucleic acid of the present disclosure comprises a nucleotide sequenceencoding a polypeptide comprising, from N-terminus to C-terminus: a) anIg Fc polypeptide; b) a proteolytically cleavable linker; c) an HCV E1polypeptide; d) two amino acids from the N-terminus of E2; and e) avariant HCV E2 polypeptide comprising, in order from N-terminus toC-terminus: i) one or more T-cell epitopes from an HCV polypeptide otherthan E1 or E2; and ii) an HCV E2 polypeptide.

In any of the above-described embodiments, the nucleic acid can beincluded in a recombinant expression vector, as described above.Suitable proteolytically cleavable linkers are described above.

V. Host Cells

The present disclosure provides genetically modified host cells, wherethe genetically modified host cells are genetically modified with anucleic acid or recombinant expression vector of the present disclosure.

Suitable host cells include eukaryotic cells, such as yeast cells,insect cells, and mammalian cells. In some cases, the host cell is acell of a mammalian cell line. Suitable mammalian cell lines includehuman cell lines, non-human primate cell lines, rodent (e.g., mouse,rat) cell lines, and the like. Suitable mammalian cell lines include,but are not limited to, HeLa cells (e.g., American Type CultureCollection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61,CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells(e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No.CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No.CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonickidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, MRC4 fibroblastcells, and the like.

Methods for introduction of nucleic acids into host cells include, forexample, transformation, electroporation, conjugation, calcium phosphatemethods and the like. The method for transfer can be selected so as toprovide for stable expression of the introduced polypeptide-encodingnucleic acid. The polypeptide-encoding nucleic acid can be provided asan inheritable episomal element (e.g., a plasmid) or can begenomically-integrated.

Methods of Producing a Variant E2 Polypeptide, a Variant E1 Polypeptide,or an E1/E2 Heterodimer

E1, E2, variant E2, variant E1, and E1/E2 polypeptides can be producedusing any suitable method, including recombinant and non-recombinantmethods (e.g., chemical synthesis).

Where a polypeptide is chemically synthesized, the synthesis may proceedvia liquid phase or solid-phase. Solid-phase peptide synthesis (SPPS)allows the incorporation of unnatural amino acids and/or peptide/proteinbackbone modification. Various forms of SPPS, such as Fmoc and Boc, areavailable for synthesizing polypeptides. Details of the chemicalsynthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev. MedChem. 6:3-10 and Camarero J A et al. 2005 Protein Pept Lett. 12:723-8).

Where a polypeptide is produced using recombinant techniques, thepolypeptide may be produced as an intracellular protein or as ansecreted protein, using any suitable construct and any suitable hostcell, which can be a prokaryotic or eukaryotic cell, such as a bacterial(e.g., Escherichia coli) cell or a yeast host cell, respectively. Otherexamples of eukaryotic cells that may be used as host cells includeinsect cells, mammalian cells, filamentous fungi, and plant cells.Suitable yeast cells include, e.g., Saccharomyces cerevisiae and Pichia(e.g., Pichia pastoris).

Suitable mammalian cells include human cell lines, non-human primatecell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitablemammalian cell lines include, but are not limited to, HeLa cells (e.g.,American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g.,ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573),Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHKcells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells,COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No.CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2cells, MRC5 cells (ATCC No. CCL-171), and the like. Where mammalian hostcells are used, such host cells may include human cells (e.g., HeLa,293, H9 and Jurkat cells); mouse cells (e.g., NIH3T3, L cells, and C127cells); primate cells (e.g., Cos 1, Cos 7 and CV1); MRC4 cells; andhamster cells (e.g., Chinese hamster ovary (CHO) cells).

A variety of host-vector systems suitable for the expression of apolypeptide may be employed according to standard procedures known inthe art. See, e.g., Sambrook et al., 1989 Current Protocols in MolecularBiology Cold Spring Harbor Press, New York; Ausubel et al. 1995 CurrentProtocols in Molecular Biology, Eds. Wiley and Sons; “ProteinExpression: A Practical Approach” (1999) S. J. Higgins and B. D. James,eds., Oxford University Press; “Protein Expression in Mammalian Cells:Methods and Protocols (Methods in Molecular Biology)” (2012) James L.Hartley, ed., Humana Press; and “Production of Recombinant Proteins”(2005) Gerd Gellisen, ed., Wiley-VCH. Methods for introduction ofnucleic acids into host cells include, for example, transformation,electroporation, conjugation, calcium phosphate methods and the like.The method for transfer can be selected so as to provide for stableexpression of the introduced polypeptide-encoding nucleic acid. Thepolypeptide-encoding nucleic acid can be provided as an inheritableepisomal element (e.g., a plasmid) or can be genomically-integrated. Avariety of appropriate vectors for use in production of a peptide ofinterest are available commercially.

Suitable expression vectors include, but are not limited to, baculovirusvectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids,bacterial artificial chromosomes, viral vectors (e.g. viral vectorsbased on vaccinia virus, poliovirus, adenovirus, adeno-associated virus,SV40, herpes simplex virus, HIV-based lentivirus vectors, murineleukemia virus (MVL)-based gamma retrovirus vectors, and the like),P1-based artificial chromosomes, yeast plasmids, yeast artificialchromosomes, and any other vectors specific for specific hosts ofinterest (such as E. coli, mammalian cells, insect cells, or yeastcells).

An E1 polypeptide, an E2 polypeptide, a variant E2 polypeptide, avariant E1 polypeptide, or an E1/E2 heterodimer can be produced byintroducing a recombinant expression vector comprising a nucleotidesequence encoding the E1 polypeptide, E2 polypeptide, variant E2polypeptide, variant E1 polypeptide, or E1/E2 heterodimer into anappropriate host cell, where the host cell produces the encoded E1polypeptide, E2 polypeptide, variant E2 polypeptide, variant E1polypeptide, or E1/E2 heterodimer. In the expression vector, apolynucleotide comprising a nucleotide sequence(s) encoding the E1polypeptide, E2 polypeptide, variant E2 polypeptide, variant E1polypeptide, or E1/E2 heterodimer is linked to a regulatory sequence asappropriate to obtain the desired expression properties. Theseregulatory sequences can include promoters, enhancers, terminators,operators, repressors, and inducers. The promoters can be regulated orconstitutive. Expression vectors generally have convenient restrictionsites located near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding a protein of interest. A selectablemarker operative in the expression host cell may be present.

In some cases, the E1 polypeptide, E2 polypeptide, variant E2polypeptide, variant E1 polypeptide, or E1/E2 heterodimer is encoded ina recombinant expression vector suitable for expression in a eukaryotichost cell (e.g., an insect cell; a yeast cell; a mammalian host cell,such as CHO cells, HeLa cells, 293 cells, MRC5 cells, etc.). In somecases, a recombinant expression vector comprises a nucleotide sequenceencoding E1 and E2 polypeptides (which may be wild-type or variant) as asingle polypeptide chain; the recombinant expression vector isintroduced into a eukaryotic host cell to generate a geneticallymodified host cell. In some cases, E1 and variant E2 polypeptides areinitially produced as a single polypeptide chain, which is cleaved inthe endoplasmic reticulum (ER) of the genetically modified host cell toproduce separate E1 and variant E2 polypeptides. The separate E1 andvariant E2 polypeptides can form a heterodimer (e.g., a non-covalentlylinked heterodimer) in the ER. In some cases, variant E1 and E2polypeptides are initially produced as a single polypeptide chain, whichis cleaved in the ER of the genetically modified host cell to produceseparate variant E1 and E2 polypeptides. The separate variant E1 and E2polypeptides can form a heterodimer (e.g., a non-covalently linkedheterodimer) in the ER The E1/E2 heterodimer can be isolated from thegenetically modified host cell by, e.g., lysis using a non-ionicdetergent, or using a freeze-thaw method. See, e.g., Frey et al. (2010)Vaccine 28:6367. The E1/E2 heterodimer can be purified from a celllysate and/or cell culture medium using any of a variety of methods,including size exclusion chromatography, affinity chromatography, andthe like, or combinations of such methods. In some cases, the E1/E2heterodimer is purified from cell lysate and/or cell culture mediumusing Galanthus nivalis (GNA) lectin affinity chromatography. In somecases, the E1/E2 heterodimer is purified from a cell lysate. In somecases, the E1/E2 heterodimer is secreted from a cell and is purifiedfrom the cell culture medium. Suitable methods that can be used forpurifying an E1/E2 heterodimer are described in, e.g., U.S. Pat. Nos.6,121,020; 6,274,148; and Mazzocca et al. (2005) J. Biol. Chem.280:11329. For example, in some cases, an E1/E2 heterodimer can beprepared in a method comprising cell disruption and debris removal bymicrofiltration, followed by purification using three subsequentchromatographic steps: lectin affinity chromatography, hydroxyapatitechromatography, and ion exchange chromatography.

Alternatively, the E1 and variant E2 polypeptides, or variant E1 and E2polypeptides, can be encoded on separate recombinant expression vectors;and produced in a cell (e.g., the same host cell or separate host cells)as separate polypeptides.

If full-length E1 and variant E2 polypeptides are expressed in aeukaryotic host cell, the E1 and variant E2 polypeptides remain bound tothe endoplasmic reticulum (ER) membrane as asialoglycoproteins. If theE1 and variant E2 polypeptides have C-terminal truncations, such thatthe C-terminal transmembrane regions are removed, the truncatedpolypeptides are secreted and can acquire complex glycans such as sialicacid. Removal of approximately amino acids 660-746 of E2, or amino acids715-746 of E2, and removal of approximately amino acids 330-383 of E1,results in secretion of E2 and E1 from a eukaryotic host cell. If E1 andvariant E2 are co-expressed in the same eukaryotic host cell asfull-length polypeptides, they remain in the lumen of the ER as aheterodimer.

In some cases, an E2 polypeptide suitable for use in a variant E2polypeptide, or suitable for use in an E1/E2 heterodimer together with avariant E1 polypeptide, lacks a transmembrane region. For example, insome cases, an E2 polypeptide suitable for use in a variant E2polypeptide, or suitable for use in an E1/E2 heterodimer together with avariant E1 polypeptide, comprises amino acids 384-659, and lacks aminoacids 660-746 of a naturally-occurring E2 polypeptide; and may bereferred to as “E2 ectodomain polypeptide.” For example, in some cases,an E2 polypeptide suitable for use in a variant E2 polypeptide, orsuitable for use in an E1/E2 heterodimer together with a variant E1polypeptide, comprises amino acids 384-659, lacks amino acids 660-746 ofa naturally-occurring E2 polypeptide, and has a length of 276 aminoacids.

In some cases, an E1 polypeptide suitable for use in a variant E1polypeptide, or suitable for use in an E1/E2 heterodimer together with avariant E2 polypeptide, lacks a transmembrane region. For example, insome cases, an E1 polypeptide suitable for use in a variant E1polypeptide, or suitable for use in an E1/E2 heterodimer together with avariant E2 polypeptide, comprises amino acids 191-329, and lacks aminoacids 330-383 of a naturally-occurring E1 polypeptide; and may bereferred to as an “E1 ectodomain polypeptide.” For example, in somecases, an E1 polypeptide suitable for use in a variant E1 polypeptide,or suitable for use in an E1/E2 heterodimer together with a variant E2polypeptide, comprises amino acids 191-329, lacks amino acids 330-383 ofa naturally-occurring E1 polypeptide, and has a length of 139 aminoacids.

After production in a host cell, an E1 polypeptide, an E2 polypeptide, avariant E2 polypeptide, a variant E1 polypeptide, or an E1/E2heterodimer (e.g., as separate polypeptides or as a heterodimer) can bepurified from the host cell. Methods of purification of recombinantlyproduced polypeptides from a host cell are known in the art and include,e.g., detergent lysis (e.g., with a non-ionic detergent) or freeze-thawlysis, followed by one or more of size exclusion column chromatography,high performance liquid chromatography, affinity chromatography, and thelike.

In some cases, an E1/E2 heterodimer of the present disclosure isproduced using a method comprising: A) culturing a genetically modifiedeukaryotic host cell that is genetically modified with a nucleic acid(e.g., recombinant expression vector) comprising a nucleotide sequenceencoding an E1/E2 polypeptide that comprises, in order from N-terminusto C-terminus: a) a signal peptide that directs the E1/E2 polypeptide tothe ER following translation of the E1/E2 polypeptide; b) an HCV E1polypeptide; c) an Ig Fc region; d) a proteolytically cleavable linker;and e) a variant E2 polypeptide of the present disclosure, where thevariant HCV E2 polypeptide comprises: i) an HCV E2 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide); B) contacting alysate of the cultured genetically modified eukaryotic host cell with asolid support comprising an Ig Fc binding moiety, generating animmobilized heterodimer comprising the HCV E1 polypeptide and a fusionpolypeptide comprising: a) the Ig Fc; b) the proteolytically cleavablelinker; and c) the variant E2 polypeptide; C) contacting the immobilizedheterodimer with an enzyme that cleaves the proteolytically cleavablelinker, thereby releasing the heterodimer; and D) collecting thereleased heterodimer.

In some cases, an E1/E2 heterodimer of the present disclosure isproduced using a method comprising: A) culturing a genetically modifiedeukaryotic host cell that is genetically modified with a nucleic acid(e.g., recombinant expression vector) comprising a nucleotide sequenceencoding an E1/E2 polypeptide that comprises, in order from N-terminusto C-terminus: a) a signal peptide that directs the E1/E2 polypeptide tothe ER following translation of the E1/E2 polypeptide; b) an HCV E1polypeptide; c) an Ig Fc region; d) a proteolytically cleavable linker;and e) a variant E2 polypeptide of the present disclosure, where thevariant HCV E2 polypeptide comprises: i) an HCV E2 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopespresent in an HCV NS3 polypeptide; B) contacting a lysate of thecultured genetically modified eukaryotic host cell with a solid supportcomprising an Ig Fc binding moiety, generating an immobilizedheterodimer comprising the HCV E1 polypeptide and a fusion polypeptidecomprising: a) the Ig Fc; b) the proteolytically cleavable linker; andc) the variant E2 polypeptide; C) contacting the immobilized heterodimerwith an enzyme that cleaves the proteolytically cleavable linker,thereby releasing the heterodimer; and D) collecting the releasedheterodimer.

In some cases, an E1/E2 heterodimer of the present disclosure isproduced using a method comprising: A) culturing a genetically modifiedeukaryotic host cell that is genetically modified with a nucleic acid(e.g., recombinant expression vector) comprising a nucleotide sequenceencoding an E1/E2 polypeptide that comprises, in order from N-terminusto C-terminus: a) a signal peptide that directs the E1/E2 polypeptide tothe ER following translation of the E1/E2 polypeptide; b) an HCV E1polypeptide; c) an Ig Fc region; d) a proteolytically cleavable linkercomprising the amino acid sequence LEVLFQGP (SEQ ID NO:5), and having alength of from 8 amino acids to 15 amino acids; and e) a variant E2polypeptide of the present disclosure, where the variant HCV E2polypeptide comprises: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide); B) contacting a lysate of thecultured genetically modified eukaryotic host cell with a solid supportcomprising an Ig Fc binding moiety, generating an immobilizedheterodimer comprising the HCV E1 polypeptide and a fusion polypeptidecomprising: a) the Ig Fc; b) the proteolytically cleavable linker; andc) the variant E2 polypeptide; C) contacting the immobilized heterodimerwith an enzyme (e.g., a rhinovirus 3C protease) that cleaves theproteolytically cleavable linker, thereby releasing the heterodimer; andD) collecting the released heterodimer.

In some cases, an E1/E2 heterodimer of the present disclosure isproduced using a method comprising: A) culturing a genetically modifiedeukaryotic host cell that is genetically modified with a nucleic acid(e.g., recombinant expression vector) comprising a nucleotide sequenceencoding an E1/E2 polypeptide that comprises, in order from N-terminusto C-terminus: a) a signal peptide that directs the E1/E2 polypeptide tothe ER following translation of the E1/E2 polypeptide; b) an HCV E1polypeptide; c) an Ig Fc region; d) a proteolytically cleavable linkercomprising the amino acid sequence LEVLFQGP (SEQ ID NO:5), and having alength of from 8 amino acids to 15 amino acids; and e) a variant E2polypeptide of the present disclosure, where the variant HCV E2polypeptide comprises: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide that comprises one or more T-cell epitopes (e.g., one ormore T cell epitopes present in an HCV polypeptide other than an HCV E1polypeptide or an HCV E2 polypeptide); B) contacting a lysate of thecultured genetically modified eukaryotic host cell with a solid supportcomprising an Ig Fc binding moiety, generating an immobilizedheterodimer comprising the HCV E1 polypeptide and a fusion polypeptidecomprising: a) the Ig Fc; b) the proteolytically cleavable linker; andc) the variant E2 polypeptide; C) contacting the immobilized heterodimerwith an enzyme (e.g., a fusion polypeptide comprising aglutathione-S-transferase and a human rhinovirus 3C protease (GST-HRV3Cprotease)) that cleaves the proteolytically cleavable linker, therebyreleasing the E1E2 heterodimer; and D) collecting the released E1E2heterodimer. In some cases, a solution comprising the released E1E2heterodimer is applied to glutathione immobilized on a solid support, toremove the GST-HRV3C protease. For example, a solution comprising thereleased heterodimer can be applied to a glutathione-Sepharose 4Bcolumn, where the GST-HRV3C binds to the glutathione-Sepharose 4B; theflow-through (unbound material) comprises the released E1E2 heterodimer.In some cases, the released E1E2 heterodimer is further subjected tohydroxyapatite chromatography. Hydroxyapatite chromatography can becarried out as described in, e.g., Mazzocca et al. (2005) J. Biol. Chem.280:11329.

Suitable Ig Fc binding moieties include, but are not limited to, ProteinA (Graille et al. (2000) Proc. Nat. Acad. Sci. USA 97:5399); Protein G(Sjöbring et al. (1991) J. Biol. Chem. 266:399); and a Protein A/Gfusion polypeptide (Eliasson et al. (1988) J. Biol. Chem. 263:4323).

The Ig Fc binding moiety can be immobilized onto a solid support, wherethe solid support can be of any of a variety of forms, e.g., a bead, amagnetic bead, a plate, and the like. The solid support can be made ofany of a variety of materials, including, but not limited to,polystyrene, agarose, polyesters, polyethylene, and the like.

FIG. 5A and FIG. 8A-8B demonstrate a purification scheme and purifiedproteins, respectively, for Fc-tagged E1E2 constructs. As an alternativeto Fc, an affinity tag such as, e.g., polyhistidine (e.g., (His)₆),glutathione-S-transferase (GST), calmodulin-binding peptide (CBP),Streptavidin-binding peptide (SBP), Strep-tag II, FLAG (e.g., DYKDDDDK(SEQ ID NO:52), hemagglutinin (HA) (e.g., YPYDVPDYA (SEQ ID NO:53),c-myc T7 ((e.g., EQKLISEEDL; SEQ ID NO:54), Glu-Glu, and the like, canbe used. (Wood D. 2014. Current Opinion in Structural Biology 26 54-61;Kimple M E et al. 2013. Current Protocols in Protein Science9.9.1-9.9.23). Other suitable affinity tags include, e.g.,starch-binding domain (SBD); and Flag-Acidic-Target Tag (FATT). See,e.g., Wood D. 2014. Current Opinion in Structural Biology 26 54-61).

One or more additional purification steps can be carried out. Forexample, a solution comprising the released heterodimer, produced asdescribed above, can be subjected to size exclusion chromatography,hydroxyapatite chromatography, and the like. Hydroxyapatitechromatography can be carried out as described in, e.g., Mazzocca et al.(2005) J. Biol. Chem. 280:11329.

An E1/E2 heterodimer of the present disclosure can be purified such thatthe E1/E2 heterodimer is at least 60% pure, at least 65% pure, at least70% pure, at least 75% pure, at least 80% pure, at least 85% pure, atleast 90% pure, at least 95% pure, at least 98% pure, at least 99% pure,or greater than 99% pure.

Immunogenic Compositions

The present disclosure provides an immunogenic composition comprising anE1/E2 heterodimer of the present disclosure. The present disclosure alsoprovides an immunogenic composition comprising a variant HCV E2polypeptide of the present disclosure. The present disclosure alsoprovides an immunogenic composition comprising a variant HCV E1polypeptide of the present disclosure.

E1/E2 Heterodimers Comprising an HCV E1 Polypeptide and a Variant E2Polypeptide

In some cases, an immunogenic composition of the present disclosureincludes an E1/E2 heterodimer of the present disclosure, the E1/E2heterodimer comprising an HCV E1 polypeptide and a variant E2polypeptide of the present disclosure. The E1 polypeptide and variant E2polypeptide present in a subject immunogenic composition may be presentin the composition as a covalently or non-covalently linked heterodimer.The E1 and variant E2 polypeptides can be present in the composition asa single polypeptide chain, or can be present as two separatepolypeptide chains (which may or may not be covalently linked via adisulfide bond).

The E1 and variant E2 polypeptides are isolated, and can be purified. Insome cases, a subject immunogenic composition comprises E1 and variantE2 polypeptides, where the polypeptides (or mixtures of E1 and variantE2 polypeptides) are at least 60% pure, at least 65% pure, at least 70%pure, at least 75% pure, at least 80% pure, at least 85% pure, at least90% pure, at least 95% pure, at least 98% pure, at least 99% pure, orgreater than 99% pure. In some embodiments, a subject immunogeniccomposition does not include any other polypeptides (e.g. no other HCVpolypeptides) other than HCV E1 and variant HCV E2 polypeptides.

In some cases, where an immunogenic composition of the presentdisclosure includes an HCV E1 polypeptide and a variant E2 polypeptideof the present disclosure, the ratio of variant E2 polypeptide to HCV E1polypeptide is in a range of from about 2:1 to 1:1, e.g., from about 2:1to 1.5:1, or from 1.5:1 to 1:1. In some cases, where an immunogeniccomposition of the present disclosure includes an HCV E1 polypeptide anda variant E2 polypeptide of the present disclosure, the molar ratio ofvariant E2 polypeptide to HCV E1 polypeptide is in a range of from about1:1 to 1.5:1, from 1.5:1 to 2:1, from 2:1 to 3:1, from 3:1 to 4:1, from4:1 to 6:1, or from 6:1 to 8:1.

An immunogenic composition of the present disclosure can comprise:

1) an E1/E2 heterodimer comprising: a) a variant E2 polypeptide of thepresent disclosure, where the variant HCV E2 polypeptide comprises: i)an HCV genotype 1 E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 1 E1polypeptide;

2) an E1/E2 heterodimer comprising: a) a variant E2 polypeptide of thepresent disclosure, where the variant HCV E2 polypeptide comprises: i)an HCV genotype 1 E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 2 E1polypeptide;

3) an E1/E2 heterodimer comprising: a) a variant E2 polypeptide of thepresent disclosure, where the variant HCV E2 polypeptide comprises: i)an HCV genotype 1 E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 3 E1polypeptide;

4) an E1/E2 heterodimer comprising: a) a variant E2 polypeptide of thepresent disclosure, where the variant HCV E2 polypeptide comprises: i)an HCV genotype 2 E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 1 E1polypeptide;

5) an E1/E2 heterodimer comprising: a) a variant E2 polypeptide of thepresent disclosure, where the variant HCV E2 polypeptide comprises: i)an HCV genotype 2 E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 2 E1polypeptide;

6) an E1/E2 heterodimer comprising: a) a variant E2 polypeptide of thepresent disclosure, where the variant HCV E2 polypeptide comprises: i)an HCV genotype 2 E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 3 E1polypeptide;

7) an E1/E2 heterodimer comprising: a) a variant E2 polypeptide of thepresent disclosure, where the variant HCV E2 polypeptide comprises: i)an HCV genotype 3 E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 1 E1polypeptide;

8) an E1/E2 heterodimer comprising: a) a variant E2 polypeptide of thepresent disclosure, where the variant HCV E2 polypeptide comprises: i)an HCV genotype 3 E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 2 E1polypeptide; or

9) an E1/E2 heterodimer comprising: a) a variant E2 polypeptide of thepresent disclosure, where the variant HCV E2 polypeptide comprises: i)an HCV genotype 3 E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 3 E1polypeptide.

An immunogenic composition of the present disclosure can comprise:

1) a first E1/E2 heterodimer and a second E1/E2 heterodimer, where: A)the first E1/E2 heterodimer comprises: a) a variant E2 polypeptide ofthe present disclosure, where the variant HCV E2 polypeptide comprises:i) an HCV genotype 1A E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 1AE1 polypeptide; and B) the second E1/E2 heterodimer comprises: a) avariant E2 polypeptide of the present disclosure, where the variant HCVE2 polypeptide comprises: i) an HCV genotype 2A E2 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 2A E1 polypeptide;

2) a first E1/E2 heterodimer and a second E1/E2 heterodimer, where: A)the first E1/E2 heterodimer comprises: a) a variant E2 polypeptide ofthe present disclosure, where the variant HCV E2 polypeptide comprises:i) an HCV genotype 1A E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 1AE1 polypeptide; and B) the second E1/E2 heterodimer comprises: a) avariant E2 polypeptide of the present disclosure, where the variant HCVE2 polypeptide comprises: i) an HCV genotype 3A E2 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 3A E1 polypeptide; or

3) a first E1/E2 heterodimer and a second E1/E2 heterodimer, where: A)the first E1/E2 heterodimer comprises: a) a variant E2 polypeptide ofthe present disclosure, where the variant HCV E2 polypeptide comprises:i) an HCV genotype 2A E2 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 2AE1 polypeptide; and B) the second E1/E2 heterodimer comprises: a) avariant E2 polypeptide of the present disclosure, where the variant HCVE2 polypeptide comprises: i) an HCV genotype 3A E2 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 3A E1 polypeptide.

An immunogenic composition of the present disclosure can comprise:

1) a first E1/E2 heterodimer, a second E1/E2 heterodimer, and a thirdE1/E2 heterodimer where: A) the first E1/E2 heterodimer comprises: a) avariant E2 polypeptide of the present disclosure, where the variant HCVE2 polypeptide comprises: i) an HCV genotype 1A E2 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 1A E1 polypeptide; B) the second E1/E2heterodimer comprises: a) a variant E2 polypeptide of the presentdisclosure, where the variant HCV E2 polypeptide comprises: i) an HCVgenotype 2A E2 polypeptide; and ii) a heterologous polypeptide thatcomprises one or more T-cell epitopes (e.g., one or more T cell epitopespresent in an HCV polypeptide other than an HCV E1 polypeptide or an HCVE2 polypeptide; e.g., one or more T-cell epitopes present in an HCV NS3polypeptide), as described above; and b) an HCV genotype 2A E1polypeptide; and C) the third E1/E2 heterodimer comprises: a) a variantE2 polypeptide of the present disclosure, where the variant HCV E2polypeptide comprises: i) an HCV genotype 3A E2 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 3A E1 polypeptide;

2) a first E1/E2 heterodimer, a second E1/E2 heterodimer, and a thirdE1/E2 heterodimer where: A) the first E1/E2 heterodimer comprises: a) avariant E2 polypeptide of the present disclosure, where the variant HCVE2 polypeptide comprises: i) an HCV genotype 1A E2 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 1A E1 polypeptide; B) the second E1/E2heterodimer comprises: a) a variant E2 polypeptide of the presentdisclosure, where the variant HCV E2 polypeptide comprises: i) an HCVgenotype 2A E2 polypeptide; and ii) a heterologous polypeptide thatcomprises one or more T-cell epitopes (e.g., one or more T cell epitopespresent in an HCV polypeptide other than an HCV E1 polypeptide or an HCVE2 polypeptide; e.g., one or more T-cell epitopes present in an HCV NS3polypeptide), as described above; and b) an HCV genotype 2A E1polypeptide; and C) the third E1/E2 heterodimer comprises: a) a variantE2 polypeptide of the present disclosure, where the variant HCV E2polypeptide comprises: i) an HCV genotype 7A E2 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 7A E1 polypeptide; or

3) a first E1/E2 heterodimer, a second E1/E2 heterodimer, and a thirdE1/E2 heterodimer where: A) the first E1/E2 heterodimer comprises: a) avariant E2 polypeptide of the present disclosure, where the variant HCVE2 polypeptide comprises: i) an HCV genotype 1A E2 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 1A E1 polypeptide; B) the second E1/E2heterodimer comprises: a) a variant E2 polypeptide of the presentdisclosure, where the variant HCV E2 polypeptide comprises: i) an HCVgenotype 3A E2 polypeptide; and ii) a heterologous polypeptide thatcomprises one or more T-cell epitopes (e.g., one or more T cell epitopespresent in an HCV polypeptide other than an HCV E1 polypeptide or an HCVE2 polypeptide; e.g., one or more T-cell epitopes present in an HCV NS3polypeptide), as described above; and b) an HCV genotype 3A E1polypeptide; and C) the third E1/E2 heterodimer comprises: a) a variantE2 polypeptide of the present disclosure, where the variant HCV E2polypeptide comprises: i) an HCV genotype 7A E2 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 7A E1 polypeptide.

Other combinations of E1/E2 heterodimers are also possible.

E1/E2 Heterodimers Comprising an HCV E2 Polypeptide and a Variant E1Polypeptide

In some cases, an immunogenic composition of the present disclosureincludes an E1/E2 heterodimer of the present disclosure, the E1/E2heterodimer comprising an HCV E2 polypeptide and a variant E1polypeptide of the present disclosure. The E2 polypeptide and thevariant E1 polypeptide present in a subject immunogenic composition maybe present in the composition as a covalently or non-covalently linkedheterodimer. The E2 and variant E1 polypeptides can be present in thecomposition as a single polypeptide chain, or can be present as twoseparate polypeptide chains (which may or may not be covalently linkedvia a disulfide bond).

The variant E1 polypeptides and the E2 polypeptides are isolated, andcan be purified. In some cases, a subject immunogenic compositioncomprises variant E1 polypeptides and E2 polypeptides, where thepolypeptides (or mixtures of variant E1 polypeptides and E2polypeptides) are at least 60% pure, at least 65% pure, at least 70%pure, at least 75% pure, at least 80% pure, at least 85% pure, at least90% pure, at least 95% pure, at least 98% pure, at least 99% pure, orgreater than 99% pure. In some embodiments, a subject immunogeniccomposition does not include any other polypeptides (e.g. no other HCVpolypeptides) other than HCV E1 and variant HCV E2 polypeptides.

In some cases, where an immunogenic composition of the presentdisclosure includes an HCV E2 polypeptide and a variant E1 polypeptideof the present disclosure, the ratio of variant HCV E2 polypeptide tovariant E1 polypeptide is in a range of from about 2:1 to 1:1, e.g.,from about 2:1 to 1.5:1, or from 1.5:1 to 1:1. In some cases, where animmunogenic composition of the present disclosure includes an HCV E2polypeptide and a variant E1 of the present disclosure, the molar ratioof variant HCV E2 polypeptide to variant E1 polypeptide is in a range offrom about 1:1 to 1.5:1, from 1.5:1 to 2:1, from 2:1 to 3:1, from 3:1 to4:1, from 4:1 to 6:1, or from 6:1 to 8:1.

An immunogenic composition of the present disclosure can comprise:

1) an E1/E2 heterodimer comprising: a) a variant E1 polypeptide of thepresent disclosure, where the variant HCV E1 polypeptide comprises: i)an HCV genotype 1 E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 1 E2polypeptide;

2) an E1/E2 heterodimer comprising: a) a variant E1 polypeptide of thepresent disclosure, where the variant HCV E1 polypeptide comprises: i)an HCV genotype 1 E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 2 E2polypeptide;

3) an E1/E2 heterodimer comprising: a) a variant E1 polypeptide of thepresent disclosure, where the variant HCV E1 polypeptide comprises: i)an HCV genotype 1 E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 3 E2polypeptide;

4) an E1/E2 heterodimer comprising: a) a variant E1 polypeptide of thepresent disclosure, where the variant HCV E1 polypeptide comprises: i)an HCV genotype 2 E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 1 E2polypeptide;

5) an E1/E2 heterodimer comprising: a) a variant E1 polypeptide of thepresent disclosure, where the variant HCV E1 polypeptide comprises: i)an HCV genotype 2 E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 2 E2polypeptide;

6) an E1/E2 heterodimer comprising: a) a variant E1 polypeptide of thepresent disclosure, where the variant HCV E1 polypeptide comprises: i)an HCV genotype 2 E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 3 E2polypeptide;

7) an E1/E2 heterodimer comprising: a) a variant E1 polypeptide of thepresent disclosure, where the variant HCV E1 polypeptide comprises: i)an HCV genotype 3 E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 1 E2polypeptide;

8) an E1/E2 heterodimer comprising: a) a variant E1 polypeptide of thepresent disclosure, where the variant HCV E1 polypeptide comprises: i)an HCV genotype 3 E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 2 E2polypeptide; or

9) an E1/E2 heterodimer comprising: a) a variant E1 polypeptide of thepresent disclosure, where the variant HCV E1 polypeptide comprises: i)an HCV genotype 3 E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 3 E2polypeptide.

An immunogenic composition of the present disclosure can comprise:

1) a first E1/E2 heterodimer and a second E1/E2 heterodimer, where: A)the first E1/E2 heterodimer comprises: a) a variant E1 polypeptide ofthe present disclosure, where the variant HCV E1 polypeptide comprises:i) an HCV genotype 1A E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 1AE2 polypeptide; and B) the second E1/E2 heterodimer comprises: a) avariant E1 polypeptide of the present disclosure, where the variant HCVE1 polypeptide comprises: i) an HCV genotype 2A E1 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 2A E2 polypeptide;

2) a first E1/E2 heterodimer and a second E1/E2 heterodimer, where: A)the first E1/E2 heterodimer comprises: a) a variant E1 polypeptide ofthe present disclosure, where the variant HCV E1 polypeptide comprises:i) an HCV genotype 1A E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 1AE2 polypeptide; and B) the second E1/E2 heterodimer comprises: a) avariant E1 polypeptide of the present disclosure, where the variant HCVE1 polypeptide comprises: i) an HCV genotype 3A E1 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 3A E2 polypeptide; or

3) a first E1/E2 heterodimer and a second E1/E2 heterodimer, where: A)the first E1/E2 heterodimer comprises: a) a variant E1 polypeptide ofthe present disclosure, where the variant HCV E1 polypeptide comprises:i) an HCV genotype 2A E1 polypeptide; and ii) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide; e.g., one or more T-cell epitopes present inan HCV NS3 polypeptide), as described above; and b) an HCV genotype 2AE2 polypeptide; and B) the second E1/E2 heterodimer comprises: a) avariant E1 polypeptide of the present disclosure, where the variant HCVE1 polypeptide comprises: i) an HCV genotype 3A E1 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 3A E2 polypeptide.

An immunogenic composition of the present disclosure can comprise:

1) a first E1/E2 heterodimer, a second E1/E2 heterodimer, and a thirdE1/E2 heterodimer where: A) the first E1/E2 heterodimer comprises: a) avariant E1 polypeptide of the present disclosure, where the variant HCVE1 polypeptide comprises: i) an HCV genotype 1A E1 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 1A E2 polypeptide; B) the second E1/E2heterodimer comprises: a) a variant E1 polypeptide of the presentdisclosure, where the variant HCV E1 polypeptide comprises: i) an HCVgenotype 2A E1 polypeptide; and ii) a heterologous polypeptide thatcomprises one or more T-cell epitopes (e.g., one or more T cell epitopespresent in an HCV polypeptide other than an HCV E1 polypeptide or an HCVE2 polypeptide; e.g., one or more T-cell epitopes present in an HCV NS3polypeptide), as described above; and b) an HCV genotype 2A E2polypeptide; and C) the third E1/E2 heterodimer comprises: a) a variantE1 polypeptide of the present disclosure, where the variant HCV E1polypeptide comprises: i) an HCV genotype 3A E1 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 3A E2 polypeptide;

2) a first E1/E2 heterodimer, a second E1/E2 heterodimer, and a thirdE1/E2 heterodimer where: A) the first E1/E2 heterodimer comprises: a) avariant E1 polypeptide of the present disclosure, where the variant HCVE1 polypeptide comprises: i) an HCV genotype 1A E1 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 1A E2 polypeptide; B) the second E1/E2heterodimer comprises: a) a variant E1 polypeptide of the presentdisclosure, where the variant HCV E1 polypeptide comprises: i) an HCVgenotype 2A E1 polypeptide; and ii) a heterologous polypeptide thatcomprises one or more T-cell epitopes (e.g., one or more T cell epitopespresent in an HCV polypeptide other than an HCV E1 polypeptide or an HCVE2 polypeptide; e.g., one or more T-cell epitopes present in an HCV NS3polypeptide), as described above; and b) an HCV genotype 2A E2polypeptide; and C) the third E1/E2 heterodimer comprises: a) a variantE1 polypeptide of the present disclosure, where the variant HCV E1polypeptide comprises: i) an HCV genotype 7A E1 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 7A E2 polypeptide; or

3) a first E1/E2 heterodimer, a second E1/E2 heterodimer, and a thirdE1/E2 heterodimer where: A) the first E1/E2 heterodimer comprises: a) avariant E1 polypeptide of the present disclosure, where the variant HCVE1 polypeptide comprises: i) an HCV genotype 1A E1 polypeptide; and ii)a heterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 1A E2 polypeptide; B) the second E1/E2heterodimer comprises: a) a variant E1 polypeptide of the presentdisclosure, where the variant HCV E1 polypeptide comprises: i) an HCVgenotype 3A E1 polypeptide; and ii) a heterologous polypeptide thatcomprises one or more T-cell epitopes (e.g., one or more T cell epitopespresent in an HCV polypeptide other than an HCV E1 polypeptide or an HCVE2 polypeptide; e.g., one or more T-cell epitopes present in an HCV NS3polypeptide), as described above; and b) an HCV genotype 3A E2polypeptide; and C) the third E1/E2 heterodimer comprises: a) a variantE1 polypeptide of the present disclosure, where the variant HCV E1polypeptide comprises: i) an HCV genotype 7A E1 polypeptide; and ii) aheterologous polypeptide that comprises one or more T-cell epitopes(e.g., one or more T cell epitopes present in an HCV polypeptide otherthan an HCV E1 polypeptide or an HCV E2 polypeptide; e.g., one or moreT-cell epitopes present in an HCV NS3 polypeptide), as described above;and b) an HCV genotype 7A E2 polypeptide.

Other combinations of E1/E2 heterodimers are also possible.

Formulations

HCV E1 polypeptides, HCV E2 polypeptides, variant E2 polypeptides,variant E1 polypeptides, and E1/E2 heterodimers can be formulated with apharmaceutically acceptable excipient(s) to generate a subjectimmunogenic composition. A wide variety of pharmaceutically acceptableexcipients is known in the art and need not be discussed in detailherein. Pharmaceutically acceptable excipients have been amply describedin a variety of publications, including, for example, A. Gennaro (2000)“Remington: The Science and Practice of Pharmacy”, 20th edition,Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and DrugDelivery Systems (1999) H. C. Ansel et al., eds 7th ed., Lippincott,Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.

In some embodiments, the E1 polypeptides, E2 polypeptides, variant E2polypeptides, variant E1 polypeptides, or E1/E2 heterodimers areformulated in an aqueous buffer. Suitable aqueous buffers include, butare not limited to, acetate, succinate, citrate, and phosphate buffersvarying in strengths from about 5 mM to about 100 mM. In someembodiments, the aqueous buffer includes reagents that provide for anisotonic solution. Such reagents include, but are not limited to, sodiumchloride; and sugars e.g., mannitol, dextrose, sucrose, and the like. Insome embodiments, the aqueous buffer further includes a non-ionicsurfactant such as polysorbate 20 (TWEEN®20) or polysorbate 80(TWEEN®80). For example, a formulation of E1 and variant E2 polypeptidesin an aqueous buffer can include, e.g., from about 0.01% to about 0.05%polysorbate-20 (TWEEN®20) non-ionic detergent. Optionally theformulations may further include a preservative. Suitable preservativesinclude, but are not limited to, a benzyl alcohol, phenol,chlorobutanol, benzalkonium chloride, and the like. In many cases, theformulation is stored at about 4° C. Formulations may also belyophilized, in which case they generally include cryoprotectants suchas sucrose, trehalose, lactose, maltose, mannitol, and the like.Lyophilized formulations can be stored over extended periods of time,even at ambient temperatures. In some cases, the aqueous buffer furtherincludes a non-ionic surfactant. In some cases, the aqueous bufferincludes the non-ionic surfactant Triton™X-100, e.g., 0.1% Triton™X-100.

E1 polypeptides, E2 polypeptides, variant E2 polypeptides, variant E1polypeptides, and E1/E2 heterodimers can be formulated into preparationsfor injection by dissolving, suspending or emulsifying them in anaqueous or nonaqueous solvent, such as vegetable or other similar oils,synthetic aliphatic acid glycerides, esters of higher aliphatic acids orpropylene glycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

An immunogenic composition of the present disclosure can include, e.g.,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium,carbonate, and the like. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like.

The concentration of E1 polypeptides, E2 polypeptides, variant E2polypeptides, variant E1 polypeptides, or E1/E2 heterodimers in aformulation can vary widely (e.g., from less than about 0.1% to at leastabout 2%, to as much as 20% to 50% or more by weight) and can beselected primarily based on fluid volumes, viscosities, andpatient-based factors in accordance with the particular mode ofadministration selected and the patient's needs.

The HCV polypeptide-containing formulations of the present disclosurecan be provided in the form of a solution, suspension, tablet, pill,capsule, powder, gel, cream, lotion, ointment, aerosol or the like. Itis recognized that oral administration can require protection of thecompositions from digestion. This is typically accomplished either byassociation of the composition with an agent that renders it resistantto acidic and enzymatic hydrolysis or by packaging the composition in anappropriately resistant carrier. Means of protecting from digestion arewell known in the art.

The HCV polypeptide-containing formulations of the present disclosurecan also be provided so as to enhance serum half-life of the heterodimerfollowing administration. For example, where isolated E1 polypeptides,E2 polypeptides, variant E2 polypeptides, variant E1 polypeptides, orE1/E2 heterodimers are formulated for injection, the HCV polypeptide maybe provided in a liposome formulation, prepared as a colloid, or otherconventional techniques for extending serum half-life. A variety ofmethods are available for preparing liposomes, as described in, e.g.,Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos.4,235,871, 4,501,728 and 4,837,028. The preparations may also beprovided in controlled release or slow-release forms.

Adjuvant

An immunogenic composition of the present disclosure can include anadjuvant. Examples of known suitable adjuvants that can be used inhumans include, but are not necessarily limited to, alum, aluminumphosphate, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5% w/v Tween80™, 0.5% w/v Span 85), CpG-containing nucleic acid (where the cytosineis unmethylated), QS21, MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT/CTmutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A,interleukins, and the like. For experimental animals, one can useFreund's, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dip-almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined by oneor more of measuring the amount of antibodies directed against theimmunogenic antigen or antigenic epitope thereof, measuring a cytotoxicT lymphocyte response to the antigen, and measuring a helper T cellresponse to the antigen.

Further exemplary adjuvants to enhance effectiveness of the compositioninclude, but are not limited to: (1) oil-in-water emulsion formulations(with or without other specific immunostimulating agents such as muramylpeptides (see below) or bacterial cell wall components), such as forexample (a) MF59TM (see, e.g., WO 90/14837), containing 5% Squalene,0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE)formulated into submicron particles using a microfluidizer, (b) SAF,containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymerL121, and thr-MDP either microfluidized into a submicron emulsion orvortexed to generate a larger particle size emulsion, and (c) RIBI™adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2%Squalene, 0.2% Tween 80, and one or more bacterial cell wall componentssuch as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cellwall skeleton (CWS), e.g., MPL+CWS (Detox™); (2) saponin adjuvants, suchas QS21 or Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may beused or particles generated therefrom such as ISCOMs (immunostimulatingcomplexes), which ISCOMS may be devoid of additional detergent e.g. WO00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2,IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.), interferons (e.g.gamma interferon), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454, optionally inthe substantial absence of alum when used with pneumococcal saccharidese.g. WO 00/56358; (6) combinations of 3dMPL with, for example, QS21and/or oil-in-water emulsions (see, e.g. EP-A-0835318, EP-A-0735898,EP-A-0761231); (7) oligonucleotides comprising a CpG motif containing atleast one CG dinucleotide, where the cytosine is unmethylated (see,e.g., WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495,WO 98/37919 and WO 98/52581); (8) a polyoxyethylene ether or apolyoxyethylene ester (see, e.g. WO 99/52549); (9) a polyoxyethylenesorbitan ester surfactant in combination with an octoxynol (WO 01/21207)or a polyoxyethylene alkyl ether or ester surfactant in combination withat least one additional non-ionic surfactant such as an octoxynol (WO01/21152); (10) a saponin and an immunostimulatory oligonucleotide (e.g.a CpG oligonucleotide) (WO 00/62800); (11) an immunostimulant and aparticle of metal salt (see, e.g. WO 00/23105); (12) a saponin and anoil-in-water emulsion (see e.g. WO 99/11241); (13) a saponin (e.g.QS21)+3dMPL+IM2 (optionally including a sterol) (see, e.g. WO 98/57659);(14) other substances that act as immunostimulating agents to enhancethe efficacy of the composition. Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc. Also suitable for use is Matrix-M™; Matrix-M™ is anadjuvant that comprises 40 nm nanoparticles comprising Quillajasaponins, cholesterol, and phospholipid. Adjuvants suitable foradministration to a human are of particular interest. In some cases, theadjuvant is one that enhances a CD4⁺ T helper response to the immunogen.

In some instances, the adjuvant is MF59, with or without aCpG-containing oligonucleotide. In other instances, the adjuvant isalum, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is poly(D,L-lactide-co-glycolide), with orwithout a CpG-containing oligonucleotide. In other instances, theadjuvant is MPL, with or without a CpG-containing oligonucleotide. Insome cases, the adjuvant is Matrix-M™, with or without a CpG-containingoligonucleotide. In some cases, the adjuvant is keyhole limpethemocyanin.

Methods of Inducing an Immune Response to HCV

The present disclosure provides a method of inducing an immune response(e.g., a protective immune response) to at least one HCV genotype in amammalian subject. In some cases, the methods comprise administering toan individual in need thereof an effective amount of a heterodimericpolypeptide of the present disclosure, or a composition (e.g., animmunogenic composition) comprising a heterodimeric polypeptide of thepresent disclosure. In other cases, the methods comprise administeringto an individual in need thereof an effective amount of a nucleicacid(s) (e.g., a recombinant expression vector) comprising nucleotidesequences encoding a heterodimeric polypeptide of the presentdisclosure.

An HCV immunogenic composition of the present disclosure, or a nucleicacid(s) comprising nucleotide sequences encoding a heterodimericpolypeptide of the present disclosure, is generally administered to ahuman subject who has an HCV infection or who is at risk of acquiring anHCV infection (e.g., is at greater risk than the general population ofacquiring an HCV infection) so as to prevent or at least partiallyarrest the development of disease and its complications. An amountadequate to accomplish this is defined as a “therapeutically effectivedose” or a “therapeutically effective amount.” “Prophylactic” use of asubject immunogenic composition generally refers to administration to anindividual who has not been infected with HCV. “Therapeutic” use of asubject immunogenic composition can refer to “prophylactic” use(administration to an individual who has not been infected with HCV)and/or to administration to an individual who has an HCV infection. A“therapeutically effective amount” of an immunogenic composition of thepresent disclosure, can be an amount that, when administered in one ormore doses to an individual who is not infected with HCV, is effectiveto induce an immune response in the individual to HCV. A“therapeutically effective amount” of an immunogenic composition of thepresent disclosure, can be an amount that, when administered in one ormore doses to an individual who is infected with HCV, is effective toenhance an immune response in the individual to HCV.

Amounts effective for therapeutic use will depend on, e.g., theimmunogenic composition or the nucleic acid(s) comprising nucleotidesequences encoding a heterodimeric polypeptide of the presentdisclosure, the manner of administration, the weight and general stateof health of the patient, and the judgment of the prescribing physician.Single or multiple doses of a subject immunogenic composition, or anucleic acid(s) comprising nucleotide sequences encoding a heterodimericpolypeptide of the present disclosure, can be administered depending onthe dosage and frequency required and tolerated by the patient, androute of administration.

In some cases, an effective amount (e.g., a therapeutically effectiveamount) of an HCV E1/E2 immunogenic composition of the presentdisclosure, or a nucleic acid(s) comprising nucleotide sequencesencoding a heterodimeric polypeptide of the present disclosure, is anamount that, when administered to an individual in one or more doses, iseffective to induce an antibody response (e.g., a neutralizing antibodyresponse) to HCV in the individual. For example, antibody to HCV (e.g.,extracellular HCV), and/or to an HCV-infected cell, can be induced.

An effective amount of an HCV E1/E2 immunogenic composition of thepresent disclosure, or a nucleic acid(s) comprising nucleotide sequencesencoding a heterodimeric polypeptide of the present disclosure, can bean amount that, when administered to an individual in one or more doses,is effective to induce a neutralizing antibody response to HCV of avariety of genotypes (e.g., genotype 1; genotype 3; etc.). Aneutralizing antibody response reduces binding of HCV to one or morehost receptors for HCV and inhibits entry of HCV into a cell.

In some cases, an effective amount (e.g., a therapeutically effectiveamount) of an HCV E1/E2 immunogenic composition of the presentdisclosure, or a nucleic acid(s) comprising nucleotide sequencesencoding a heterodimeric polypeptide of the present disclosure, is anamount that, when administered to an individual in one or more doses, iseffective to induce a cytotoxic T lymphocyte (CTL) response to HCV. Forexample, a CTL response to an HCV-infected cell can be induced.

In some cases, an effective amount (e.g., a therapeutically effectiveamount) of an HCV E1/E2 immunogenic composition of the presentdisclosure, or a nucleic acid(s) comprising nucleotide sequencesencoding a heterodimeric polypeptide of the present disclosure, is anamount that, when administered to an individual in one or more doses, iseffective to induce a helper T lymphocyte (e.g., CD4⁺ T cell) to HCV inan individual.

In some cases, an effective amount (e.g., a therapeutically effectiveamount) of an HCV E1/E2 immunogenic composition of the presentdisclosure, or a nucleic acid(s) comprising nucleotide sequencesencoding a heterodimeric polypeptide of the present disclosure, is anamount that, when administered to an individual in one or more doses, iseffective to induce an antibody response (e.g., a neutralizing antibodyresponse) and/or a CTL response and/or a helper T cell response to HCVgenotype 1. In some cases, an effective amount (e.g., a therapeuticallyeffective amount) of an HCV E1/E2 immunogenic composition of the presentdisclosure, or a nucleic acid(s) comprising nucleotide sequencesencoding a heterodimeric polypeptide of the present disclosure, is anamount that, when administered to an individual in one or more doses, iseffective to induce an antibody response (e.g., a neutralizing antibodyresponse) and/or a CTL response and/or a helper T cell response to HCVgenotype 3. In some cases, an effective amount (e.g., a therapeuticallyeffective amount) of an HCV E1/E2 immunogenic composition of the presentdisclosure, or a nucleic acid(s) comprising nucleotide sequencesencoding a heterodimeric polypeptide of the present disclosure, is anamount that, when administered to an individual in one or more doses, iseffective to induce an antibody response (e.g., a neutralizing antibodyresponse) and/or a CTL response and/or a helper T cell response to HCVgenotype 1 and HCV genotype 3. In some cases, an effective amount (e.g.,a therapeutically effective amount) of an HCV E1/E2 immunogeniccomposition of the present disclosure, or a nucleic acid(s) comprisingnucleotide sequences encoding a heterodimeric polypeptide of the presentdisclosure, is an amount that, when administered to an individual in oneor more doses, is effective to induce an antibody response (e.g., aneutralizing antibody response) and/or a CTL response and/or a helper Tcell response to HCV of any genotype.

An HCV E1/E2 immunogenic composition of the present disclosure, or anucleic acid(s) comprising nucleotide sequences encoding a heterodimericpolypeptide of the present disclosure, is generally administered in anamount effective to elicit an immune response, e.g., a humoral immuneresponse (e.g., an antibody response) and/or a CTL response, in themammalian subject. Effective amounts for immunization will vary, and cangenerally range from about 1 μg to 100 μg per 70 kg patient, e.g., fromabout 5 g/70 kg to about 50 μg/70 kg. Substantially higher dosages (e.g.10 mg to 100 mg or more) may be suitable in oral, nasal, or topicaladministration routes. The initial administration can be followed bybooster immunization of the same HCV E1/E2 immunogenic composition or adifferent HCV E1/E2 immunogenic composition. In some instances, asubject method of inducing an immune response involves an initialadministration of an HCV E1/E2 immunogenic composition of the presentdisclosure, followed by at least one booster, and in some instancesinvolves two or more (e.g., three, four, or five) boosters. The intervalbetween an initial administration and a booster, or between a givebooster and a subsequent booster, can be from about 1 week to about 12weeks, e.g., from about 1 week to about 2 weeks, from about 2 weeks toabout 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeksto about 8 weeks, from about 8 weeks to about 10 weeks, or from about 10weeks to about 12 weeks.

In general, immunization can be accomplished by administration of an HCVE1/E2 immunogenic composition of the present disclosure, or a nucleicacid(s) comprising nucleotide sequences encoding a heterodimericpolypeptide of the present disclosure, by any suitable route, includingadministration of the composition orally, nasally, nasopharyngeally,parenterally, enterically, gastrically, topically, transdermally,subcutaneously, intramuscularly, in tablet, solid, powdered, liquid,aerosol form, locally or systemically, with or without added excipients.Actual methods for preparing parenterally administrable compositionswill be known or apparent to those skilled in the art and are describedin more detail in such publications as Remington's PharmaceuticalScience, 15th ed., Mack Publishing Company, Easton, Pa. (1980). In someinstances, immunization is accomplished by intramuscular injection of anHCV E1/E2 immunogenic composition of the present disclosure.

In some cases, a method of the present disclosure for inducing an immuneresponse to HCV in an individual comprises administering to theindividual an effective amount of a nucleic acid(s) comprisingnucleotide sequences encoding a heterodimeric polypeptide of the presentdisclosure. In some cases, the nucleic acid is present in an expressionvector. Suitable expression vectors include, but are not limited to, areplication-defective adenovirus vector; a replication-defectivevaccinia virus vector; a lentivirus vector (e.g., a self-inactivatinglentivirus vector); a retroviral vector (e.g., a self-inactivatingretroviral vector); an adeno-associated virus vector; and the like. Insome cases, the vector is a modified vaccinia Ankara (MVA) vector, or anMVA-based vector (see, e.g., Verheust et al. (2012) Vaccine 30:2623).

In some cases, a method of the present disclosure for inducing an immuneresponse to HCV in an individual comprises administering to theindividual an effective amount of a nucleic acid(s) comprisingnucleotide sequences encoding a heterodimeric polypeptide of the presentdisclosure. In some cases, the nucleic acid is an RNA comprisingnucleotide sequences encoding a heterodimeric polypeptide of the presentdisclosure. See, e.g., Weiner (2013) Molec. Therapy 21:506; and Ulmer etal. (2012) Vaccine 30:4414. In some cases, an RNA (e.g., a single mRNAmolecule; or 2 mRNA molecules) comprising nucleotide sequences encodinga heterodimeric polypeptide of the present disclosure is formulated witha liposome. In some cases, an RNA (e.g., a single mIRNA molecule; or 2mRNA molecules) comprising nucleotide sequences encoding a heterodimericpolypeptide of the present disclosure is complexed with protamine. Insome cases, an RNA (e.g., a single mRNA molecule; or 2 mRNA molecules)comprising nucleotide sequences encoding a heterodimeric polypeptide ofthe present disclosure is complexed with1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOTAP/DOPE).

Individuals Suitable for Administration

Individuals who are suitable for administration with an HCV compositionof the present disclosure include immunologically naïve individuals(e.g., individuals who have not been infected with HCV and/or who havenot been administered with an HCV vaccine).

Individuals who are suitable for administration with an HCV compositionof the present disclosure include individuals who are at greater riskthan the general population of becoming infected with HCV, where suchindividuals include, e.g., intravenous drug users; individuals who arethe recipients, or the prospective recipients, of blood or bloodproducts from another (donor) individual(s); individuals who are therecipients, or the prospective recipients, of non-autologous cells,tissues, or organs from another (donor) individual; health care workers;emergency medical and non-medical personnel (e.g., first responders;fire fighters; emergency medical team personnel; etc.) and the like.

Individuals who are suitable for administration with an HCV compositionof the present disclosure include individuals who recently becameexposed to HCV or who recently became infected with HCV. For example, asubject immunogenic composition can be administered to an individualwithin from about 24 hours to about 48 hours, from about 48 hours toabout 1 week, or from about 1 week to about 4 weeks, following possibleor suspected exposure to HCV or following infection with HCV.

Individuals who are suitable for administration with an HCV compositionof the present disclosure include individuals who have been diagnosed ashaving an HCV infection, and include chronically infected individuals.In some cases, an individual who has been diagnosed as having an HCVinfection is treated with an anti-viral agent and a subject HCVimmunogenic composition. Suitable anti-viral agents for treating HCVinfection include, e.g., ribavirin(1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide); interferon-alpha(IFN-α) (where “IFN-α” includes IFN-α2a; IFN-α2b; IFN-α that isconjugated with poly(ethylene glycol) (“pegylated IFN-α), where thepegylated IFN-α can be pegylated IFN-α2a or pegylated IFN-α 2b); an HCVNS3 protease inhibitor (e.g., boceprevir; telaprevir); and an HCV NS5protease inhibitor. In some cases, an individual who has been diagnosedas having an HCV infection is treated with, e.g.: 1) IFN-α+ribavirin;and a subject HCV immunogenic composition; or 2) IFN-α+ribavirin+an HCVprotease inhibitor (e.g., boceprevir or telaprevir); and a subject HCVimmunogenic composition. Suitable anti-viral agents for treating HCVinfection include Sovaldi (Sofosbuvir; a nucleotide analog thatfunctions as an NS5B polymerase inhibitor), alone or in combination withpegylated IFN-α and ribavirin.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Identification of T-Cell Epitopes in HCV NS3

Consensus sequences for various HCV genotypes were built using Geneioussoftware. This included consensus sequences for genotype HCV1a, 1b, 2a,2b, 3, 4, 5, and 6 from 224, 357, 20, 27, 30, 18, 3, and 59 differentisolates, respectively. For genotype HCV7, the only reported isolate wasused in the alignment.

To identify CD4 T cell epitopes, all the reported CD4⁺ T cell epitopesthrough Jul. 12, 2013 were extracted from the Immune Epitope Databaseand Analysis Resource (IDEB). The sequence of each epitope was enteredinto an alignment, including sequences from HCV genotypes 1a, 1b, 2a,2b, and 3, as these are common HCV genotypes around the world. More than287 epitopes that were reported in 518 studies as MHC II-restricted CD4T cells epitopes with positive response in humans for HCV wereextracted. Of those, which were grouped in 159 clusters at 70% identity,217 were located and annotated on the sequences of HCV genotypes of 1a,1b, 2a, 2b, and 3. In the case of mismatch of 1-3 amino acids in thesequence of the epitope against the HCV sequences, the epitope was stillincluded in the analysis. The excluded 70 epitopes consisted of thosethat were located on structural regions of HCV polyprotein (core, E1,E2, and P7), were repeats of other epitopes, or were not found on thesequence. After addition of all epitopes, it appeared that almost thewhole non-structural region of HCV polyprotein from residue number 941(based on the HCV1a sequence), in the middle of non-structural protein 2(NS2), to the residue number 3000, close to the end of NS5b were heavilycovered by epitopes with exception of the regions 1980-2081 (thebeginning of NS5a) and 2710-2788 (in the middle of NS5b).

Two additional analyses were performed: first, a Conservancy Analysisfor all 217 epitopes was performed using IDEB software. In one analysis,all epitopes were checked for conservation amongst all 9 HCV genotypes(HCV1a, 1b, 2a, 2b, 3, 4, 5, 6, and 7), which resulted in 6 conservedepitopes and in the second analysis, the same list of 217 epitopes wereevaluated against genotypes 1a, 1b, and 3 that resulted in 16 conservedepitopes. The partially overlapped epitopes were combined and of thecompletely overlapped epitopes, the largest one was retained. Thisresulted in a total of 11 so called “Region” that were conserved amongall 3 genotypes of HCV1a, 1b, and 3. Of these 11, 4 were conserved among9 HCV genotypes (HCV1a, 1b, 2a, 2b, 3, 4, 5, 6, and 7) and the rest wereconserved among 4 to 8 genotypes (Table 1; FIG. 10 ).

Similar to CD4 epitopes, all reported CD8 T cell epitopes through Jul.12, 2013 were extracted from the Immune Epitope Database and AnalysisResource (IDEB). A total number of 413 epitopes in 216 clusters at 70%from 1226 studies were found; however, to narrow down the list, analternative search performed based on the common HLA class-I Alleles inNorth America. According to the latest demographic statistics from 2013,the population of USA as the representative of the North Americacomprises of different ethnic groups including White (64%), Black (12%),Hispanic (16%), Asian or Pacific Islanders (5%), and others includingNatives, Alaskans, and etc. (3%). The first 30 common MHC-I alleles(frequency >˜6-7%) for each of the first four common populations in USAwere used in a search for any HCV epitopes that were reported for any ofthese individual alleles, which comprised of A*01:01, A*02:03, A*02:06,A*02:07, A*03:01, A*11:01, A*23:01, A*24:02, A*25:01, A*26:01, A*29:02,A*30:01, A*30:02, A*31:01, A*32:01, A*33:03, A*34:02, A*68:01, A*68:02,A*74:01, B*07:02, B*08:01, B*14:02, B*15:01, B*15:02, B*15:03, B*18:01,B*35:01, B*38:02, B*40:01, B*40:02, B*42:01, B*44:01, B*44:03, B*45:01,B*46:01, B*49:01, B*51:01, B*52:01, B*53:01, B*54:01, B*55:02, B*57:01,B*58:01, C*01:02, C*02:02, C*03:03, C*03:04, C*04:01, C*05:01, C*06:02,C*07:01, C*07:02, C*08:01, C*08:02, C*14:02, and C*16:01.

Using IDEB database through Jul. 12, 2013, a total of 106 MHC-I CD8 Tcell epitopes that were restricted to each of the afore-mentionedalleles were found as described in Table 2 (FIG. 11 ) with theirspecific anchor positions. The sequence of each epitope was searched andentered in an alignment including sequences from HCV genotypes 1a, 1b,and 3 as the genotypes of interest for a vaccine that will bepotentially used in North America. Of 106 epitopes, 64 were located andannotated on the sequences of HCV genotypes of 1a, 1b, and 3. In thecase of mismatch of 1-3 amino acids in the sequence of the epitopeagainst the HCV sequences, the epitope was still included in theanalysis. The excluded 42 epitopes consisted of those that were locatedon structural regions of HCV polyprotein (core, E1, E2, and P7), wererepeats of other epitopes, or were not found on the sequence.

A Conservancy Analysis for all 106 epitopes was performed using IDEBsoftware. In one analysis, all epitopes were checked for conservancyagainst nine HCV genotypes (1a, 1b, 2a, 2b, 3, 4, 5, 6, and 7) resultedin 2 conserved epitopes and in the second analysis the same list ofepitopes were evaluated against genotypes 1a, 1b, and 3 that resulted in6 conserved epitopes. The partially overlapped epitopes were combinedand of the completely overlapped epitopes, the largest one was retained.This resulted in a total of 5 conserved epitopes or combination ofepitopes (called Regions) with all to be conserved among 3 genotypes and2 to be conserved among all 9 HCV genotypes. The list of these regions(CD8-R1 to CD8-R5) is described in Table 3 (FIG. 12 ).

In addition, a Conservancy Analysis for all 413 CD8 epitopes was doneusing IDEB software. When checked for conservancy against nine HCVgenotypes (1a, 1b, 2a, 2b, 3, 4, 5, 6, and 7), 2 new conserved epitopeswere found and in the second analysis the same list of epitopes wereevaluated against genotypes 1a, 1b, and 3 that resulted in 7 newconserved epitopes. The partially overlapped epitopes were combined andof the completely overlapped epitopes, the largest one was retained.This resulted in a total of 5 new conserved epitopes or combination ofepitopes (called Regions) with all to be conserved among 3 genotypes and1 to be conserved among all 9 HCV genotypes. The list of these regions(CD8-R5 to CD8-R10) is described in Table 3 (FIG. 12 ).

According to the Immunodominancy Analysis (IDEB) and the relativelocation of conserved epitopes, as well to use a minimal sequence withwhich to extend the C-terminus of gpE2, 5 antigens were chosen based onCD4 epitopes and 4 antigens based on CD8 epitopes (Table 4; FIG.13A-13D) to be used for extension of gpE2. For each selected antigen(Ag), the number of CD4 and CD8 regions that are included is described(Table 4; FIG. 13A-13D).

The CD8-based antigens were longer, so as to include more CD8 epitopesfrom different HLAs. The focus was on CD4-based antigens (CD4-Ag-1 toCD4-Ag-5) that also include CD8 epitopes; accordingly, 4 antigens Table5; FIG. 14 ) were cloned to gpE1/gp/E2 sequence for expression andpurification and animal studies.

Example 2: Expression of Fc-Tagged E1E2 with or without T-Cell Polytope(TPx) Extensions

As shown in the schematic diagram (FIG. 5A) the nucleotide sequence forthe full-length HCV E1E2 glycoprotein was inserted downstream of thecytomegalovirus promoter (PCMV) in a mammalian cell expression vector.The E1E2 sequence is preceded by the signal peptide sequence from tissueplasminogen activator (tPA) to direct the polypeptide to the endoplasmicreticulum (ER) following translation of the polypeptide. At the Nterminus of E2, a duplication of amino acids 384-385 (ET) was insertedfollowed by the human IgG1 Fc tag and a PreScission Protease (PP)recognition sequence (LEVLFQGP; SEQ ID NO:5). Constructs containing aT-cell polytope (TPx; x=29, 52 or 100 amino acids) have this sequenceinserted downstream of the PP recognition site. After translation of theE1E2 polypeptide and entry into the ER, host-derived signal peptidase(SP) cleaves the signal sequences in the tPA signal peptide as well asthe C-terminus of E1, resulting in E1 and E2 polypeptides. Forpurification of E1E2 heterodimers, PreScission Protease (PP) can beadded to the Fc-tagged E1E2 protein (immobilized on Protein A or ProteinG affinity resins) to remove the Fc tag from the N terminus of the E2polypeptide. This strategy can be applied to HCV E1E2 glycoproteins ofdifferent genotypes (FIGS. 6A-7B and 7A-8B).

As shown in the schematic diagram (FIG. 5A) the nucleotide sequence forthe full-length HCV E1E2 glycoprotein was inserted downstream of thecytomegalovirus promoter (PCMV) in a mammalian cell expression vector.The E1E2 sequence is preceded by the signal peptide sequence from tissueplasminogen activator (tPA) to direct the polypeptide to the endoplasmicreticulum (ER) following translation of the polypeptide. At the Nterminus of E2, a duplication of amino acids 384-385 relative to theparticular genotype (e.g.: ET addition for H77; GenBank NP_671941) wasinserted followed by the human IgG1 Fc tag and a PreScission Protease(PP) recognition sequence (LEVLFQGP; SEQ ID NO:5). Constructs containinga T-cell polytope (TPx; x=29, 52 or 100 amino acids; e.g., TP29, TP52,and TP100 as set forth in FIG. 14A-14D) have this sequence inserteddownstream of the PP recognition site. After translation of the E1E2polypeptide and entry into the ER, host-derived signal peptidase (SP)cleaves the signal sequences in the tPA signal peptide as well as theC-terminus of E1, resulting in E1 and E2 polypeptides. For purificationof E1E2 heterodimers, PreScission Protease (PP) can be added to theFc-tagged E1E2 protein (immobilized on Protein A or Protein G affinityresins) to remove the Fc tag from the N terminus of the E2 polypeptide.This strategy can be applied to HCV E1E2 glycoproteins of differentgenotypes (FIGS. 6A-7B and 7A-8B).

FIG. 5A-5B. Schematic representation of Fc-tagged and untagged E1E2expression constructs and polypeptide processing. The E1E2 polypeptideis expressed under the control of the CMV promoter (PcMv) and includesthe signal sequence from tissue plasminogen activator (tPA). Insertionsites are shown for representative HCV E1E2 sequences: H77 (GenBankNP_671941) and Alberta isolate Avi1a129 (genotype 1A), JFH1 (GenbankAB047639; genotype 2A), S52 (Genbank ADF97232.1; genotype 3a), Albertaisolate Avi3a177 (genotype 3A), and isolate QC69 (Genbank: ABN05226.1;genotype 7A). Sizes of the polypeptide regions are shown at the top(aa=amino acids). (A) Fc tagged E1E2: At the N-terminus of E2, aduplication of the E2 N-terminal amino acids respective to theparticular genotype (eg: ET addition for H77; GenBank NP_671941) isinserted followed by the human IgG1 Fc tag (hu IgG1 Fc) and aPreScission Protease (PP) recognition sequence (LEVLFQGP; SEQ ID NO:5).Constructs containing a T-cell polytope (TPx; where x is, e.g., 25, 52,or 100 amino acids) have this polytope sequence inserted C-terminal ofthe PP recognition site. Following expression of the polypeptide, signalpeptidase (SP) cleavages result in the downstream E1 and E2 polypeptidesshown. The E1 and E2 polypeptides interact to form a heterodimer. Forpurification purposes, the Fc tagged E1E2 is immobilized on Protein A orProtein G resin and digested with PreScission Protease (PP) (cleavagebetween Q and G in the LEVLFQGP (SEQ ID NO:5) sequence) to release theuntagged E1E2 heterodimer. (B) Untagged E1E2: At the N-terminus of E2, aduplication of the E2 N-terminal amino acids respective to theparticular genotype (eg: ET addition for H77; GenBank NP_671941) isinserted followed by a T-cell polytope (TPx; where x is, e.g., 25, 52,or 100 amino acids). Following expression of the polypeptide, signalpeptidase (SP) cleavages result in the downstream E1 and E2 polypeptidesshown. The E1 and E2 polypeptides interact to form a heterodimer. Theuntagged E1E2 is purified using Galanthus nivalis lectin agarose (GNA)chromatography. The duplicated E2 N-terminal residues (shown: ETaddition for H77; GenBank NP_671941) and TPx are retained in thepurified E1E2.

FIG. 6A-6B. Alignment of the Fc-tagged E1-E2 polypeptide (with orwithout TPx extension) for H77 and Alberta isolate Avi1a129 (genotype1A). The amino acid sequence for the coding region of thetPa-E1-Fc-PP-TPx-E2 construct (as diagrammed in FIG. 5A-5B) for theAlberta isolate (Avi1a129) and H77 (GenBank NP_671941) was aligned usingGeneious software v5.6.4. (*) denotes the insertion of the duplicatedN-terminal E2 residues that precede the Fc-PP-TPx tag (Avi1a129: QT andH77: ET).

FIG. 7A-7B. Alignment of the Fc-tagged E1-E2 polypeptide (with orwithout TPx extension) for S52 (Genbank ADF97232.1) and Alberta isolateAvi3a177 (genotype 3A). The amino acid sequence for the coding region ofthe tPA-E1-Fc-PP-TPx-E2 construct (as diagrammed in FIG. 5A-5B) for theAlberta isolate (Avi3a177) and S52 was aligned using Geneious softwarev5.6.4. (*) denotes the insertion of the duplicated N-terminal E2residues that precede the Fc-PP-TPx tag (ET for both Avi3a177 and S52).

In FIG. 6A-6B and FIG. 7A-7B, TP29 has the sequenceAIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:1); TP52 has the sequenceAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:2); andTP100 has the sequenceVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:4).

Methods for purification of Fc-tagged E1E2 with or without T-cellpolytope (TPx) extensions from CHO cell extracts.

As shown in FIG. 8A-8B, the following purification strategy results incapture of the Fc-tagged E1E2 glycoproteins (wild type or T cellpolytope (TPx) extensions) by Protein G Sepharose chromatography medium,tag removal by PreScission Protease and the isolation of E1E2heterodimers.

CHO cells stably expressing the Fc-tagged E1E2 expression vectors werepelleted, washed twice in sterile phosphate buffered saline (PBS) andfrozen at −80° C. Frozen cell pellets were homogenized in lysis buffer(20 mM TRIS, 100 mM NaCl, 1 mM EDTA, 2% TX-100 pH 7.5) containingprotease inhibitor cocktail (Calbiochem; 539134) by pipetting andincubated on ice for 30 minutes. Cell debris was removed bycentrifugation for 15 min at 12000 g 4° C. using a JA25.5 rotor andAvanti J-26 XPI centrifuge (Beckman Coulter). Soluble cell extract(supernatant fraction) was filtered using a Stericup vacuum-drivenfiltration unit (Millipore SCGPU01RE).

Filtered cell extracts were bound to Protein G Sepharose 4 Fast Flow(GE; 17-0618-02) in batch mode (1 ml Protein G Sepharose: 1 g CHO cellextract in 40-50 ml volume) for 2 hours at 4° C. with gentle rotation.The Protein G Sepharose resin was sedimented by centrifugation at 200 gfor 5 min in an Allegra X-15R swinging bucket centrifuge (BeckmanCoulter). Unbound cell extract was removed and the Protein G Sepharosewas washed in 15 ml final volume with wash buffer (20 mM TRIS, 150 mMNaCl, 0.5% TX-100 pH 7.5) for 10 minutes at 4° C. with gentle rotation.The Protein G Sepharose was sedimented by centrifugation at 200 g for 5min and the wash step repeated two more times. The resin was resuspendedto a final volume of 5m in digestion buffer (20 mM HEPES, 250 mM NaCl,5% glycerol pH 7.5) containing GST-tagged PreScission Protease (GST-PP)(50 g) and incubated overnight (16-20 hours) with gentle rotation at 4°C.

Following incubation with GST-PP, the Protein G Sepharose wastransferred to a Poly-Prep chromatography column (Biorad; 731-1550) andthe unbound material and 2 column volume (CV) (2×1 ml) washes withdigestion buffer collected. The collected flow through was then added toa 0.5 ml Glutathione Sepharose 4B (GE; 17-0756-01) column(pre-equilibrated with 10 CV of digestion buffer) for GST-PP removal.The Glutathione Sepharose 4B flow-through and 2 CV (2×0.5m) washes withdigestion buffer was collected. The collected flow through wasconcentrated using a centrifugal filter unit (50,000 MWCO) (Amicon;UFC805024) (final volume 200-300 d) and then diluted 10-fold in HAPbuffer.

The diluted sample was applied to a hydroxyapatite type I (HAP) column;and the HAP flow through was collected. The final sample wasconcentrated using a centrifugal filter unit (50,000 molecular weightcutoff (MWCO)) (Amicon; UFC805024) to a final volume of 200-300 μl.Aliquots of the final antigen (10-20 μl) were stored at −80° C.

FIG. 8A-8B. Purification of E1E2 heterodimer from CHO cell extractsexpressing Fc-tagged E1E2. Fc-PP tagged wildtype or TP extension E1E2(E1E2 heterodimer, where E2 includes a T-cell polytope) proteins fromCHO cell extracts were immobilized on Protein-G Sepharose 4 Fast Flow,and digested with GST-PreScission protease (GST-PP) to remove the Fctag. Following the digestion with GST-PP and release of untagged E1E2,GST-PP was removed by Glutathione Sepharose 4B. The untagged E1E2protein concentrate was then applied to a HAP column. The HAP flowthrough containing the final E1E2 heterodimer was collected and samplesloaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) gel. After electrophoresis, the gel was stained, or blottedthen probed with antibodies. (A) Western blot with anti-E1 (A4) andanti-E2 (H52) monoclonal antibodies (mAbs) (0.5 g loaded per lane). (B)Colloidal Coomassie Brilliant Blue G250 stained gel (2 g loaded perlane). Lanes: (1) wild-type E1E2 (no TP extension); (2) E1-TP29-E2; (3)E1-TP52-E2; and (4) E1-TP100-E2. E1-TP29-E2: E1-E2 heterodimer, where E2includes a 29-amino acid T-cell polytope (“TP29”) as set forth in FIG.14A-D; E1-TP52-E2: E1-E2 heterodimer, where E2 includes a 52-amino acidT-cell polytope (“TP52”) as set forth in FIG. 14A-D; and E1-TP100-E2:E1-E2 heterodimer, where E2 includes a 100-amino acid T-cell polytope(“TP100”) as set forth in FIG. 14A-D.

Example 3: Analysis of E1/E2 Heterodimers

Immunization of Mice with Recombinant gpE1/gpE2 Antigens

CB6F1 mice were injected intramuscular (IM) with recombinant gpE1/gpE1antigens at day 0, 14 and 28 days. Pre-vaccination serum was collectedat day 0 at the time of the first injection and test bleeds obtained byjugular puncture at 28 days. Terminal bleeds are performed at day 42. Insome cases, an extended protocol for immunizations may be applied with afourth injection administered at day 56 and terminal bleed collected onday 70. Blood samples were centrifuged at 5000 g and serum collected andheat inactivated by incubation at 56° C. for 30 minutes. Serum sampleswere stored in aliquots at −80° C. until use.

Determination of Neutralizing Antibodies (nAb) Response to HCV fromImmunized Mice

HCVcc Assay

Methods for detecting nAb responses from the sera of immunized animalsby HCV cell culture-derived virus (HCVcc) are similar to those describedpreviously (Law, J. et al. 2013. Plos One 8(3) e59776). Human hepatomacells (Huh7.5) were plated on poly-lysine coated 96 well plates, 1 dayprior to infection. For infection, 100 TCID50 HCV cell culture-derivedvirions (HCVcc) were premixed with heat inactivated sera diluted at 1 in50 (by volume), for 1 hour at 37° C. followed by adding to cells. 12hour post-infection, the antibody-virus inoculum was replaced with freshculture media. Cells were fixed 48 hours post-infection with methanolusing previously described methods. Infection was determined byquantitation of NS5A-positive foci using mouse monoclonal NS5A antibody(9E10). Foci were detected and counted using a CTL S6 immunospotanalyzer. The percentage of neutralization was calculated in comparisonto pre-vaccination serum. The neutralization activity was calculatedusing the following formula: % neutralization=(pre−post)/pre×100% wherepre/post represent the number of NS5A-positive foci done afterincubating with either the pre- or post-vaccination sera.

HCVpp Assay

Pseudotyped viruses enclosed by HCV glycoproteins (HCVpp) were generatedby co-transfecting plasmids encoding HIV provirus expressing Luciferaseand the HCV envelope glycoproteins (gpE1/gpE2) as previously described(Hsu et al. (2003) Proc. Nat. Acad. Sci. USA 100(12) 7271-7276). On theday prior to transfection 8×10⁵ 293T cells were seeded in a 35 mm well.The following day a total of 1.5 g DNA was transfected using Fugene 6 orother transfection reagent and media replaced after 6 hpost-transfection. Supernatants containing HCVpp were harvested at 48 hand 72 h after transfection, pooled and filtered. Neutralization assayswith immunized animal sera were performed as described for the HCVecneutralization assay. Human hepatoma cells (Huh7.5) were plated onpoly-lysine coated 96-well plates. 1 day prior to infection. HCVpp werediluted 1/10 and premixed with heat inactivated sera diluted at 1 in 50(by volume) for 1 hour at 37° C. followed by adding to cells. 12 hourpost-infection, the antibody-virus inoculum was replaced with freshculture media. Cells were processed 48 hours post-infection usingBright-glo luciferase assay system (Promega). Luminescence was measuredusing an Enspire plate reader (Perkin Elmer). The neutralizationactivity was calculated using the following formula: %neutralization=(pre−post)/pre×100% where pre/post represent theluciferase activity done after incubating with either the pre- orpost-vaccination sera.

Determination of Anti-gpE2 Specific Antibodies from Immunized Mice

Detection of anti-gpE2 antibodies from immunized mice were determinedusing recombinant gpE2 enzyme-linked immunosorbent assay (ELISA).Briefly, recombinant gpE2 (amino acids 384-661) was coated to 96-wellmicrotiter plates in carbonate buffer (15 mM sodium carbonate, 35 mMsodium bicarbonate, pH 9.6) overnight at 4° C. Wells were then blockedfor one hour at room temperature with 200 μl of phosphate bufferedsaline+0.2% non-ionic detergent TWEEN-20™ (PBST) containing 5% bovineserum albumin (BSA). Wells were washed three times with 250 μl PBST and50 μl of heat inactivated sera from immunized animals added per well intriplicate in PBST for one hour at room temperature. Immunized sera wereexamined in serial dilution (eg: 1000, 2000, 4000, etc fold dilutions)and compared to either (i) pre-vaccinated sera from the same animal or(ii) a pooled pre-vaccinated serum sample from several animals. Wellswere washed three times with 250 μl PBST and incubated for one hour withhorseradish peroxidase (HRP) conjugated goat anti-mouse antibody at roomtemperature. HRP-activity was detected using peroxidase substrate andabsorbance read at 450 nm-570 nm using an Enspire plate reader (PerkinElmer).

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. One or more nucleic acids comprising nucleotidesequences encoding a heterodimeric polypeptide comprising a) a varianthepatitis C virus (HCV) E2 polypeptide comprising: i) an HCV E2polypeptide; and ii) a heterologous polypeptide comprising a T-cellepitope present in an HCV protein other than E1 and E2, wherein theheterologous polypeptide comprises an amino acid sequence having atleast 20% amino acid sequence identity to the amino acid sequenceselected from: SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:63, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:64, and SEQID NO:13; and b) an HCV E1 polypeptide.
 2. The one or more nucleic acidsof claim 1, wherein the heterologous polypeptide comprises an amino acidsequence having at least 50% amino acid sequence identity to the aminoacid sequence selected from: SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:63, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:64, and SEQ ID NO:13.
 3. The one or more nucleic acids of claim 1,wherein the heterologous polypeptide comprises an amino acid sequencehaving at least 95% amino acid sequence identity to the amino acidsequence selected from: SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:63, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:64, and SEQ ID NO:13.
 4. The one or more nucleic acids of claim 1,wherein: a) the HCV E2 polypeptide is derived from an HCV of genotype 1,2, 3, or 7; and b) the HCV E1 polypeptide is derived from an HCV ofgenotype 1, 2, 3, or
 7. 5. The one or more nucleic acids of claim 1,wherein the heterologous polypeptide has a length of from about 29 aminoacids to about 2000 amino acids.
 6. The one or more nucleic acids ofclaim 1, wherein the variant HCV E2 polypeptide comprises, in order fromN-terminus to C-terminus: i) the heterologous polypeptide and the HCV E2polypeptide; or ii) the HCV E2 polypeptide and the heterologouspolypeptide.
 7. The one or more nucleic acids of claim 1, wherein theone or more nucleic acids are one or more RNA molecules.
 8. The one ormore nucleic acids of claim 7, wherein the one or more RNA molecules areformulated with a liposome.
 9. A method of inducing an immune responseto an antigen in an individual, the method comprising administering tothe individual the one or more nucleic acids of claim
 1. 10. The methodof claim 9, wherein said administering comprises: a) administering aninitial dose of the one or more nucleic acids; and b) administering atleast one booster dose of the one or more nucleic acids.
 11. One or morenucleic acids comprising nucleotide sequences encoding a heterodimericpolypeptide comprising a) a variant hepatitis C virus (HCV) E1polypeptide comprising: i) an HCV E1 polypeptide; and ii) a heterologouspolypeptide comprising a T-cell epitope present in an HCV protein otherthan E1 and E2, wherein the heterologous polypeptide comprises an aminoacid sequence having at least 20% amino acid sequence identity to theamino acid sequence selected from: SEQ ID NO:1, SEQ ID NO:11, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:63, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:64, and SEQ ID NO:13; and b) an HCV E2 polypeptide. 12.The one or more nucleic acids of claim 11, wherein the heterologouspolypeptide comprises an amino acid sequence having at least 50% aminoacid sequence identity to the amino acid sequence selected from: SEQ IDNO:1, SEQ ID NO:11, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:63,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:64, and SEQ ID NO:13.
 13. The oneor more nucleic acids of claim 11, wherein the heterologous polypeptidecomprises an amino acid sequence having at least 95% amino acid sequenceidentity to the amino acid sequence selected from: SEQ ID NO:1, SEQ IDNO:11, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:63, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:64, and SEQ ID NO:13.
 14. The one or morenucleic acids of claim 11, wherein: a) the HCV E2 polypeptide is derivedfrom an HCV of genotype 1, 2, 3, or 7; and b) the HCV E1 polypeptide isderived from an HCV of genotype 1, 2, 3, or
 7. 15. The one or morenucleic acids of claim 11, wherein the heterologous polypeptide has alength of from about 29 amino acids to about 2000 amino acids.
 16. Theone or more nucleic acids of claim 11, wherein the variant HCV E1polypeptide comprises, in order from N-terminus to C-terminus: i) theheterologous polypeptide and the HCV E1 polypeptide; or ii) the HCV E1polypeptide and the heterologous polypeptide.
 17. The one or morenucleic acids of claim 11, wherein the one or more nucleic acids are oneor more RNA molecules.
 18. The one or more nucleic acids of claim 17,wherein the one or more RNA molecules are formulated with a liposome.19. A method of inducing an immune response to an antigen in anindividual, the method comprising administering to the individual theone or more nucleic acids of claim
 11. 20. The method of claim 19,wherein said administering comprises: a) administering an initial doseof the one or more nucleic acids; and b) administering at least onebooster dose of the one or more nucleic acids.
 21. One or more nucleicacids comprising nucleotide sequences encoding a heterodimericpolypeptide comprising a) a variant hepatitis C virus (HCV) E1polypeptide comprising: i) an HCV E1 polypeptide; and ii) a heterologouspolypeptide comprising a T-cell epitope present in an HCV protein otherthan E1 and E2, wherein the heterologous polypeptide comprises an aminoacid sequence having at least 20% amino acid sequence identity to theamino acid sequence selected from: SEQ ID NO:1, SEQ ID NO:11, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:63, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:64, and SEQ ID NO:13; and b) a variant HCV E2polypeptide comprising: i) an HCV E2 polypeptide; and ii) a heterologouspolypeptide comprising a T-cell epitope present in an HCV protein otherthan E1 and E2, wherein the heterologous polypeptide comprises an aminoacid sequence having at least 20% amino acid sequence identity to theamino acid sequence selected from: SEQ ID NO:1, SEQ ID NO:11, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:63, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:64, and SEQ ID NO:13.
 22. The one or more nucleic acidsof claim 21, wherein the heterologous polypeptide comprises an aminoacid sequence having at least 50% amino acid sequence identity to theamino acid sequence selected from: SEQ ID NO:1, SEQ ID NO:11, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:63, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:64, and SEQ ID NO:13.
 23. The one or more nucleic acidsof claim 21, wherein the heterologous polypeptide comprises an aminoacid sequence having at least 95% amino acid sequence identity to theamino acid sequence selected from: SEQ ID NO:1, SEQ ID NO:11, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:63, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:64, and SEQ ID NO:13.
 24. The one or more nucleic acidsof claim 21, wherein: a) the HCV E2 polypeptide is derived from an HCVof genotype 1, 2, 3, or 7; and b) the HCV E1 polypeptide is derived froman HCV of genotype 1, 2, 3, or
 7. 25. The one or more nucleic acids ofclaim 21, wherein the heterologous polypeptide has a length of fromabout 29 amino acids to about 2000 amino acids.
 26. The one or morenucleic acids of claim 21, wherein: a) the variant HCV E2 polypeptidecomprises, in order from N-terminus to C-terminus: i) the heterologouspolypeptide and the HCV E2 polypeptide; or ii) the HCV E2 polypeptideand the heterologous polypeptide; and b) the variant HCV E1 polypeptidecomprises, in order from N-terminus to C-terminus: i) the heterologouspolypeptide and the HCV E1 polypeptide; or ii) the HCV E1 polypeptideand the heterologous polypeptide.
 27. The one or more nucleic acids ofclaim 21, wherein the one or more nucleic acids are one or more RNAmolecules.
 28. The one or more nucleic acids of claim 27, wherein theone or more RNA molecules are formulated with a liposome.
 29. A methodof inducing an immune response to an antigen in an individual, themethod comprising administering to the individual the one or morenucleic acids of claim
 21. 30. The method of claim 29, wherein saidadministering comprises: a) administering an initial dose of the one ormore nucleic acids; and b) administering at least one booster dose ofthe one or more nucleic acids.